DE10122206A1 - Novel use of 2',5'-oligoadenylate synthetase or RNAseL polypeptides, nucleic acids encoding polypeptides, cell expressing peptides and nucleic acids or antibody for analyzing, diagnosing, or treating wound healing - Google Patents

Abstract

Use of 2',5'-oligoadenylate synthetase (I) and/or RNAseL polypeptides (II); nucleic acids (III) encoding (I) or (II); cell expressing (I), (II) or (III); or antibody (IV), for analyzing, diagnosing, preventing and/or treating, identifying active substances and producing array for the analysis in association with wound healing, is new. Use of at least one 2',5'-oligoadenylate synthetase polypeptide and/or RNAseL polypeptides of a sequence (S1) comprising 367, 473, 364, 726, 1087, and 514 amino acids, or 735 and 741 amino acids fully defined in the specification, or their functional variants; and/or nucleic acids encoding the above polypeptides, or its variants, a cell which is expressing the above polypeptides or its functional variants and/or nucleic acid, or an antibody or its fragment preferably a polyclonal or a monoclonal antibody or their fragment, for analysis, diagnosis, prevention and/or treatment, identification of active substances, and production of an array for the analysis in association with wound healing and/or its pathological conditions, where an antibody-producing organism is immunized with the synthetase polypeptide or RNAseL polypeptide or their functional variants.

Description

The invention relates to the use of or encoding polypeptides Nucleic acids of a 2'-5'-oligoadenylate synthetase and / or RNAse L for di agnosis, prevention and / or treatment of diseases of skin cells, of Wound healing and / or their pathological disorders and their use for Identification of pharmacologically active substances.

Wounds generally heal without therapeutic intervention. However, there is numerous diseases in which wound healing is pathologically impaired, such as z. B. Diabetes mellitus, arterial occlusive diseases, psoriasis (Psoria sis), Crohn's disease, epidermolysis bullosa, age-related skin changes, Contact dermatitis, atopic dermatitis or innervation disorders. Wundhei Disorders lead to delayed wound healing or chronic Wounds. These disorders can be caused by the type of wound (e.g. large area wounds, deep and mechanically stretched surgical wounds, Burns, trauma, pressure ulcers), drug treatment of patients (e.g. with corticoids) but also caused by the type of disease itself become. So suffer z. B. 25% of patients with type II diabetes often have chronic ulcers ("diabetic foot"), of which about half are elaborate statio require natural treatments and ultimately heal poorly. The diabetic Foot causes more hospital stays than any other diabetes-related Complication. The number of these cases in type I and II diabetes is increasing reefs and represents 2.5% of all hospital admissions. Wounds also heal worse with increasing age of the patients. Often there is also an acceleration  supply of the natural wound healing process desirable to z. B. the danger of bacterial infections or to reduce patient downtime.

Even after wound closure, there may be further disorders. While wounds of fetal skin heal without scarring, it happens after ver in the postnatal period always lead to scarring, which is often a major pose cosmetic problem. For patients with extensive burns quality of life can also be dramatically affected, especially since scarred ter skin also the appendages, such as hair follicles, sweat and sebum glands absence. With appropriate genetic disposition, it can also become keloids come, hypertrophic scars that overgrow the surrounding skin.

The process of wound healing requires complex, coordinated processes interactions and interactions of different cell types. In the wound healing process and one differentiates the following steps: the blood coagulation in the area of the wound, the Inflammatory cell recruitment, re-epithelialization, formation of Granulation tissue as well as the restructuring of matrix. The exact reactions pattern of the cell types involved during the phases of proliferation, the Migration, matrix synthesis and contraction are just like the regulati on genes such as B. growth factors, receptors and matrix proteins, little known so far.

So far, little satisfactory therapies have been developed to help with To be able to intervene in wound healing disorders. Established forms of therapy restrict themselves to physical support for wound healing (e.g. bandages, Compresses, gels) or the transplantation of skin tissues, grown Skin cells and / or matrix proteins. In recent years there have been growth factors have been tried to improve wound healing, but without the conven to significantly improve tional therapy. The diagnosis of Wundhei lung disorders is based on poorly meaningful optical analyzes of the skin,  since a deeper understanding of gene regulation during wound healing so far is missing.

Surprisingly, it has now been shown that a usable 2'-5'- Oligoadenylate synthetase plays an essential role in wound healing and its morbid disorders and skin disorders, and that therefore poly peptides according to SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 9 to SEQ ID No. 10 or these coding nucleic acids of a 2'-5'-oligoadenylate synthetase and / or its direct effector RNAse L for use in diagnosis, Prevention and / or treatment of diseases of skin cells, of wounds healing and / or their pathological disorders and for the identification of pharmacologically active substances are suitable.

A nucleic acid of the 2'-5'-oligoadenylates which can be used according to the invention Synthetase polypeptides according to SEQ ID No. 1 were isolated from cDNA libraries made from intact and wounded skin. The cDNAs selected that have different frequencies in well-healing in ver had wounds that were treated poorly with dexamethasone. This was done with the help of subtractive hybridization (Diatchenko et al., 1996, Proc. Natl. Acad. Sci. USA 93: 6025-30). It was shown that the 2'-5'-Oligoadenylate synthetase in wounds from with the glucocorticoid Dexa animals treated with methasone was expressed significantly more than in untreated animals Wounds.

After the primary identification of a gene, it is necessary to make the dermatitis expression and wound healing specific expression by another method to confirm. This was done using "TaqMan Analysis". With these methods de the amount of 2'-5'-oligoadenylate synthetase (2-5 OAS) mRNA in tissues determined from various wound healing states and in psoriasis (Examples 2 until 6). It could be demonstrated that the expression in Wundhei  lung is regulated differentially. In addition, in venous ulcers, one sweat ren wound healing disorder, a deficiency of 2-5 OAS mRNA can be demonstrated. Furthermore, an analysis of the individual biopsies of psoriasis patients could significantly increased amount of 2-5 OAS mRNA with regard to healthy intact skin be measured. This shows that an incorrect regulation of expression and / or Activity of a 2-5 OAS and / or its effector to cause serious wound can cause healing disorders and skin diseases.

The object is therefore achieved by the use of one or more rerer 2'-5'-oligoadenylate synthetase and / or RNAse L polypeptides according to one of SEQ ID No. 1 to SEQ ID No. 4 and SEQ ID No. 9 to SEQ ID No. 12 or functional variants thereof and / or nucleic acids encoding them or Va Rianten thereof, or a 2'-5'-oligoadenylate synthetase and / or RNAse L. Polypeptide according to one of SEQ ID No. 1 to SEQ ID No. 4 and SEQ ID No. 9 to SEQ ID No. 12 or functional variants thereof or this encoding nuc cell expressing linseed acids or variants thereof for diagnosis, treatment, and / or prevention of skin cell disorders, wound healing and / or pathological disorders and their use for the identification of dissolved pharmacologically active substances.

Various forms of the 2'-5'-oligoadenylates exist in human cells Synthetase, namely small, medium and large proteins, the former two can in turn be present in different isoforms by splicing (Re boulliat and Hovanessian, 1999, Journal of Interferon and Cytokine Research, 19: 295-300). The small proteins, p40 (SEQ ID No. 3) and p46 contain one individual catalytic unit and are referred to as OAS1. They are in vivo as Tetramer before. The OAS2 isoforms p67 and p70 (SEQ ID No. 4) contain 2 ka talytic units per polypeptide and are available as dimers. Still exists an OAS3 (SEQ ID No. 9) with 3 catalytic units, which as a monomer lies. In the mouse, OAS 1 (SEQ ID No. 1) also exists in two splice isofor  men. In addition to these polypeptides with proven catalytic activity exis animals in mouse and human OAS-like proteins (SEQ ID No. 2 and SEQ ID No. 10), which is characterized by interferon inducibility and homology to catalytic Mark unit.

2'-5'-oligoadenylate synthetase and its effector RNAse L are central enzymes me of the so-called 2-5A system, which has long been part of an antiviral Systems is known in living beings (e.g. Williams, 1979, Nature; 282: 582-586; Chebath, 1987, Nature, 330: 587-588). The amount of the 2'-5'-oligoadenylates Syn thetase can be induced by interferons (e.g. Baglioni et al., 1979, Bio chemistry, 18: 1765-1770). For activation in vitro, dsRNA is also used or ssRNA with a certain secondary structure, as z. B. in the 5'- untranslated region of the HIV virus occurs (Jacobs and Langland, 1996, Virology, 219: 339-349; Maitra et al., 1994, Virology; 204: 823-827). After Akti The ATP is converted to 2'-5'-linked oligoadenyla ten of the general formula pppA (2'-5'A) n, n ≧ 2, which under the expression 2-5A be summarized. 2-5A then directly activates the RNAse L effector, one Endoribonuclease that breaks down RNA after activation. This justifies the antivi ral effect of this enzyme: through the presence of genomic viral dsRNA molecules or dsRNA intermediates in the replication of viral Ge The 2-5 OAS is activated, thus the production of 2-5A, to activate the RNAse L and finally to destroy or prevent it replication of the virus (Rebouillat and Hovanessian, 1999, Journal of Interfe Ron and Cytokine Research, 19: 295-300). This was, for example, for the EMCV virus detected (Williams et al., 1979, supra). About the antiviral The enzyme system 2-5 OAS / RNAse L also has an effect on cell prolifera tion and differentiation (Hovanessian and Wood, 1980, Virology, 101: 81-90; Rysiecki et al., 1989, J. Interferon Res., 9: 649-657). So different stu serve to conclude that 2-5 OAS is important for growth control or has an antitumor effect (Creasy et al., 1983, Mol. Cell Biol., 3: 780-786; Rimoldi et  al., 1990, Exp. Cell Res., 191: 76-82). Correlations between 2-5 OAS and growth competence, proliferation or differentiation in various cell types have been detected (Zullo et al., 1985, Cell, 43: 793-800; Ma or et al., 1990, Differentiation, 44: 18-24; Birnbaum et al., 1993, Differentiation, 45: 138-145). In addition to viral diseases, the role of 2-5 OAS in Diseases examined in those linked to viral diseases is suspected, namely type I diabetes and the "chronic fatigue syndrome". act Regulation could actually follow in connection with these diseases (Bonnevie-Nielsen et al., 1991, J. Interferon. Res., 11: 255-260; Suhadolnik et al., 1994, In vivo, 8: 599-604). While on the pathogenesis of dia betes I no clear evidence is available in the case of "Chronic Fatigue Syndrome "markedly upregulated 2-5 OAS / RNAse L systems in patients demonstrated. A connection with wound healing or its pathological disturbances However, skin cells or diseases have not yet been proven sen or suggested.

In general, the analysis of differentially expressed genes in tissues is included clearly more errors in the form of false positive clones than the Analy se of cell culture systems. This problem cannot be solved by using of a defined cell culture system can be avoided, because existing, simple Cell culture systems do not understand the complexity of wound healing processes in tissue can simulate sufficiently.

The problem is particularly with the skin, which consists of a variety of ver different cell types. In addition, the process of wound healing is one highly complicated process, the temporal and spatial changes cellular Processes such as proliferation and differentiation in the different cells types includes. The approach, not just the complex cell system skin, but beyond the physiological process of wound healing and even various very early stages of wound healing at the level of differentially expressed genes  For a specialist, researching is therefore not a promising strategy. The Success of the screening was essential due to these difficulties depending on the choice of experimental parameters. While the used Methods (e.g. subtractive hybridization) are standard methods Grid investigation and verification strategy through the well thought out and defi The choice of parameters itself is already inventive. For example, the Time of biopsy critical for the success of the raster examination: Wound healing disorders and skin diseases are often associated with disorders Cell proliferation and cell migration. These processes become one day initiated after the wound, which is why an analysis of the molecular processes before this point in time would provide little information about the processes taking place for normal wound healing is essential. On the other hand, it changes in the course of wound healing later than one day after the wound composition of cell types in the wound strongly. This can lead to a differential expression of a particular gene in the wound was measured which is not based on altered expression in the cells, but only on the different cell composition. This shows that the choice of The day of the biopsy is decisive for the success of the raster examination affected. Despite the defined parameters, an overrepresentation of Observed genes that are differentially expressed during wound healing, which is unsuitable for use in wound healing or for skin diseases are suitable. These genes include, for example, genes that are responsible for Pri market metabolism, such as glycolysis, citrate cycle, gluconeogenesis and respiration encode chain, but also genes that code for ribosomal proteins, e.g. B. L41 and S20. It was therefore necessary to identify the 2'- 5'-oligoadenylate synthetase gene as a gene relevant to wound healing dominant.

There are also enormous variations in the wound condition at the time of a possible biopsy of the patient on first contact with the doctor. Therefore became  Identification of the nucleic acids described above an animal model ver applies. BALB / c mice were wounded and at different times Wound biopsies taken. This procedure has the advantage of being the edge conditions such as genetic background, type of wound, time of biopsy etc. can be checked precisely and only then a reproducible analysis of the Ge Allow nexpression. Even under the defined mouse conditions other methodological problems such as redundancy of the analyzed clones and Underrepresentation of poorly expressed genes that can identify relevant genes complicate.

Skin diseases, wound healing and their pathological disorders in the For the purposes of the invention, a distinction must be drawn from skin diseases which ent cell development and cell differentiation, especially of Skin cancer. In the latter disease, individual cells are transformed len, which is thereby uncontrolled, autonomous, d. H. of interactions with others Cell types begin to proliferate in isolation and thereby the pathological changes passed on to the daughter cells. So it's a disease those with a loss of interactions, such as cell-cell adhesion and is accompanied by typical cell properties. On the other hand, there are diseases in the sense of the invention on disorders of skin cells in their physiological Context. The development of skin diseases in the sense of the invention are due to a variety of factors. So play z. B. in the case of psoriasis, probably both genetic predispositions and malfunctions of the T cells, fibroblasts and keratinocytes play a role (see e.g. Nair et al., 1997; Hum. Molec. Genet. 6: 1349-1356; Gottlieb et al., 1995, Nat. Med. 1: 442-447; Saiag et al., 1985, Science, 230: 669-672; Pittelkow, 1998, in Roenigk 1998: 225- 246). The course of wound healing can also be varied by various endogenous ones and modulated exogenous factors. Even small disturbances in the interactions between the different cell types of the dermis and epidermis itself, however interactions with other tissues and organs such as the blood vessel  system, the nervous system and connective tissue, can lead to disturbed wound lead to healing followed by scarring. Infections, old people tion, diseases such as diabetes and immune diseases as well as vitamin men gel impair the wound healing process. Similarly complex interactions are also for other skin diseases such as vitiligo and atopic dermatitis described. This distinguishes the skin diseases from cancer diseases of these organs. Preferred examples of skin diseases are pso riasis, eczema, especially atopic eczema, pigmentation disorders of the skin, in particular vitiligo and hair growth or hair metabolism disorders mus.

Examples of impaired wound healing are wounds in diabetic patients and Al alcoholic wounds infected with microorganisms, ischemic wounds and Wounds from patients with poor circulation and venous congestion. Especially preferred poorly healing wounds are diabetic, neuropathic, venous and arterial ulcers, especially venous ulcers.

The autonomous nature of the cancers is also evident on therapeuti level. In the case of non-metastatic tumors, cancer can be surgically treat is. This physical treatment option is possible because between interactions between tumor cells and the surrounding cells and tissues take place so that the patient can be cured by simple excision of the tumor can, while this is not possible with skin diseases in the sense of the invention - The pathological disorders of the cell-cell and / or tissue-tissue interaction can not be remedied by cutting out the affected skin. That the compared diseases are diseases that one fundamentally different mechanism, it becomes clear when one compares the therapeutic approaches. With cancer and sick therapy associated with degenerate cell proliferation the killing of rapidly growing cells e.g. B. directed by means of cytostatics. This  toxic substances prevent the growth of actively proliferating cells while cells are not affected in the G0 phase of the cell cycle. Hinge gen is aimed at treating skin cell diseases in the sense of the Erfin modulation of the interactions between the different cell types, such as B. by influencing migration, proliferation and differentiation individual cell types. Skin diseases in the sense of the invention cannot can be cured by general inactivation of proliferating cells. The metho dische approach to identify the nucleic acid used in the invention ren that are involved in wound healing and / or processes of skin diseases in the The meaning of the invention is significantly different from methods that are involved Suitable are to identify nucleic acids that are involved in processes of the cancer crane kung are involved. The latter can be differentially expressed by analysis Genes of the cancer-affected cell type can be identified. Aim of the assay rather, it is the present invention by comparing the expressions on to identify genes in sick and healthy tissue biopsies that are linked to the complex processes of skin diseases and / or wound healing and / or their pathological disorders are involved. To identify this process would be unsuitable for cancer-relevant genes.

The term "functional variants" of a polypeptide in the sense of the present Invention encompasses polypeptides which, for example, ver used polypeptides during illness, especially skin diseases, or in the case of regenerative processes of the skin, but particularly in the case of wound heat disorders, are regulated. Functional variants include, for example se also polypeptides encoded by a nucleic acid derived from non- skin-specific tissue, e.g. B. embryonic tissue can be isolated, but after Expression in a cell involved in wound healing or skin disease have the designated functions.  

Functional variants in the sense of the present invention are also 2'-5'- Oligoadenylate synthetase and RNAse L polypeptides, which have sequence homology, in particular a sequence identity of approximately 70%, preferably approximately 80%, in particular especially about 90%, especially about 95% of the polypeptide with the amino acid sequence according to one of SEQ ID No. 1 to SEQ ID No. 4 and SEQ ID No. 9 to SEQ ID # 12. Examples of such functional variants are accordingly polypeptides homologous to a polypeptide which can be used according to the invention, which from organisms other than human or mouse, preferably from non-human mammals such as B. monkeys, pigs and rats. Other examples of functional variants are polypeptides by different alleles of the gene, in different individuals or in different Organs of an organism are encoded. Particularly preferred examples are Splice isoforms of 2 '-5'-oligoadenylate synthetase.

"Sequence identity" means the agreement (=% positive) between two sequences, which in the case of polypeptide sequences for example using BlastP 2.0.1 and in the case of polynucleotide sequences, for example with BLASTN 2.0.14 is determined, with the filter = off (Altschul et al., 1997, Nucleic Acids Res., 25: 3389-3402). To determine the similarity between For example, two polypeptides BlastP 2.0.1 is used, the Filter = off is set and BLOSUM is 62.

Functional variants of the polypeptide can also be parts of the invention used 2'-5'-oligoadenylate synthetase or RNAse L polypeptide polypep tids with at least 6 amino acids in length, preferably with at least 8 ami non-acidic length, in particular with at least 12 amino acid length. by are also N- and / or C-terminal and / or internal deletions of the inventions polypeptide used according to the invention in the range from about 1-60, preferably from about 1-30, especially about 1-15, especially about 1-5 amino acids. at for example, the first amino acid methionine may be missing without the function  of the polypeptide is changed significantly. Furthermore, a post-translational Modification, for example myristoylation, is absent without activity of the enzyme is significantly changed (Samantha et al., 1983, J. Biol. Chem, 255: 9807 to 9813).

The term "coding nucleic acid" refers to a DNA sequence that for an isolable bioactive 2'-5'-oligoadenylate synthetase according to the invention and / or RNAse L polypeptide or a precursor, e.g. B. with a signal sequence, coded. The polypeptide can be a full length or any part of a sequence the coding sequence can be encoded as long as the specific, e.g. enzymatic activity is retained.

It is known that changes in the sequence of those described above Nucleic acids can be present, for example by the degeneration of the genetic codes, or that non-translated sequences, for example, on the 5'- and / or 3 'end of the nucleic acid can be attached without the Ak activity is changed significantly. You can also use the ones described below Modifications are carried out. This invention therefore also includes so-called called "variants" of the nucleic acids described above.

"Variants" of the nucleic acids are understood to mean all DNA sequences that are complementary to a DNA sequence that are under stringent conditions hybridize with the reference sequence and have essentially the same activity have like the polypeptide encoded by the reference sequence.

The activity of a 2'-5'-oligoadenylate synthesis which can be used according to the invention tase consists in the dsRNA-dependent conversion of ATP to 2-5A. Assays for This activity is determined in Player and Torrence (1998, Pharmacol. Ther., 78: 55-113).  

The activity of an RNAse L which can be used according to the invention consists in the 2-5A dependent degradation of RNA and the 2-5A binding. A compilation Suitable assays can be found in Player and Torrence (1998, Pharmacol. Ther., 78: 55-113).

Under "stringent hybridization conditions" such conditions are too understand, where a hybridization for example at 60 ° C in 2.5 × SSC Buffer followed by multiple washes at 37 ° C in a lower buffer concentration takes place and remains stable.

Variants of the nucleic acid can also be parts of those used according to the invention Nucleic acid with at least 8 nucleotides in length, preferably with at least 18 nucleotides in length, in particular with at least 24 nucleotides in length, particularly preferably with at least 30 nucleotides, preferably with be at least 42 nucleotides.

For example, the term "regulation" means the increase or decrease understood the amount of polypeptide or nucleic acid encoding it, wherein this change, for example, at the transcriptional or translational level can take place.

The 2'-5'-oligoadenylate synthetase and RNAse L which can be used according to the invention Polypeptides can also be characterized in that they are synthetic getting produced. All or part of the polypeptide can thus be added game synthesized using classic synthesis (Merrifield technique) the. Parts of the polypeptides according to the invention are particularly suitable for Ge antisera, with the help of suitable gene expression banks can be searched in order to further functional variants of the invention appropriate polypeptide to arrive. So p69 / p71 and OAS3 were used  monoclonal antibodies isolated (Hovanessian et al., 1987, EMBO J, 6: 1273-1280; Hovanessian et al., 1988, J. Biol. Chem, 263: 4945-4949).

The nucs which can be used according to the invention are preferably linseed acids around DNA or RNA, preferably a DNA, especially a dop fur-stranded DNA. Furthermore, the sequence of the nucleic acids can thereby be characterized as having at least one intron and / or a polyA sequence having. The nucleic acids used according to the invention can also be in the form their antisense sequence.

For the expression of the relevant 2'-5'-oligoadenylate synthetase or RNAse L A double-stranded DNA is generally preferred, the gene for the DNA region encoding polypeptide is particularly preferred. This area be starts with the first in a Kozak sequence (Kozak, 1987, Nucleic. Acids Res. 15: 8125-48) start codon (ATG) to the next stop codon (TAG, TGA or TAA), which is in the same reading frame as the ATG.

Another use of the nucleic acid sequences according to the invention is Construction of anti-sense oligonucleotides (Zheng and Kemeny, 1995, Clin. Exp. Immunol. 100: 380-2; Nellen and Lichtenstein, 1993, Trends Biochem. Sci. 18: 419-23; Stein, 1992, Leukemia 6: 967-74) and / or ribozymes (Amarzguioui, et al. 1998, Cell. Mol. Life Sci. 54: 1175-202; Vaish, et al., 1998, Nucleic Acids Res. 26: 5237-42; Persidis, 1997, Nat. Biotechnol. 15: 921-2; Couture and Stinch comb, 1996, Trends Genet. 12: 510-5). With anti-sense oligonucleotides you can the stability of nucleic acids are reduced and / or the translation of Nucleic acids are inhibited. So can be used by the invention extension of the nucleic acid sequences, for example, the expression of the genes in cells are reduced both in vivo and in vitro. antisense Oligonucleotides and ribozymes can therefore be used as therapeutic agents. This strategy is also suitable for skin, epidermal and dermal, for example  Cells, especially when the antisense oligonucleotides come with liposomes plexed (Smyth et al., 1997, J. Invest. Dermatol. 108: 523-6; White et al., 1999, J. Invest. Dermatol. 112: 699-705; White et al., 1999, J. Invest. Dermatol. 112: 887-92). For use as a probe or as an "antisense" oligonucleotide single-stranded DNA or RNA is preferred.

A nucleic acid can also be used to carry out the invention that has been manufactured synthetically. Thus, the ver used nucleic acid, for example, chemically based on that in Table 1 DNA sequences written and / or based on the in these tables also described protein sequences using the genetic code z. B. can be synthesized according to the phosphotriester method (see e.g. Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584, No. 4).

Oligonucleotides are usually quickly replaced by endo- or exonucleases, in particular by DNases and RNases occurring in the cell. It is therefore advantageous to modify the nucleic acid in order to use it against the Stabilize degradation, so that a high concentration over a long period the nucleic acid is retained in the cell (Beigelman et al., 1995, Nuc leic Acids Res. 23: 3989-94; Dudycz, 1995, WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116). Typically, a sol stabilization by introducing one or more internucleotide Phosphorus groups or by introducing one or more non-phosphorus Internucleotides can be obtained.

Suitable modified internucleotides are described in Uhlmann and Peymann (1990 Chem. Rev. 90, 544) (see also Beigelman et al., 1995 Nucleic Acids Res. 23: 3989-94; Dudycz, 1995, WO 95/11910; Macadam et al., 1998, WO 98/37240; Reese et al., 1997, WO 97/29116). Modified internucleotide Phosphate residues and / or non-phosphorus bridges in a nucleic acid  one of the uses according to the invention can be used for example methylphosphonate, phosphorothioate, phosphoramidate, phosphoro dithioate, phosphate ester, while non-phosphorus internucleotide analogs, at for example siloxane bridges, carbonate bridges, carboxymethyl esters, acetami data bridges and / or thioether bridges included. It is also intended that this modification the shelf life of a pharmaceutical composition, which can be used in one of the uses according to the invention, ver repaired.

In a further embodiment of the use according to the invention, the The nucleic acids described above in a vector, preferably in an egg A "shuttle" vector. Phagemid, cosmid, expression vector or gentherapeu table effective vector included. Furthermore, the above can be described NEN nucleic acids in "knock-out" gene constructs or expression cassettes be included.

A vector which is effective in gene therapy contains wound or skin specific regulatory sequences that are functionally related to the preceding written nucleic acid are connected.

The expression vectors can be prokaryotic or eukaryotic expressions be vectors. Examples of prokaryotic expression vectors are for the Ex pression in E coli z. B. the vectors pGEM or pUC derivatives and for eukaryotic cal expression vectors for expression in Saccharomyces cerevisiae z. B. the vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768), for expression in insect cells e.g. B. Baculovirus Vectors as disclosed in EP-B1-0 127 839 or EP-B1-0 549 721, and for Expression in mammalian cells e.g. B. the vectors Rc / CMV and Rc / RSV or SV40- Vectors, all of which are commonly available.  

In general, the expression vectors also contain for the respective cell suitable promoters, e.g. B. the trp promoter for expression in E. coli (see e.g. EP-B1-0 154 133), the Met 25, GAL 1 or ADH2 promoter for the Expression in yeast (Russel et al. (1983), J. Biol. Chem. 258, 2674-2682; Mum berg, supra), the baculovirus polyhedrin promoter, for expression in insect cells (see e.g. 13. EP-B1-0 127 839). For expression in mammalian cells For example, promoters are suitable that have a constitutive, regulatable, ge web-specific, cell cycle-specific or metabolically specific expression in Allow eukaryotic cells. Adjustable elements according to the present Invention are promoters, activator sequences, enhancers, silencers and / or Repressor.

Example of suitable regulatable elements, the constitutive expression in Euka Allow ryonten are promoters recognized by the RNA polymerase III become or viral promoters, CMV enhancers, CMV promoters, SV40- Promoter or LTR promoters e.g. B. from MMTV (mouse mammary tumor virus; Lee et al. (1981) Nature 214, 228-232) and other viral promoter and assets Gate sequences derived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV or HIV.

Examples of regulatable elements that regulate expression in eukaryotes enable, are the tetracycline operator in combination with an equivalent repressor (Gossen M. et al. (1994) Curr. Opin. Biotechnol. 5, 516-20).

The expression of wound healing or skin diseases is preferably carried out relevant genes under the control of tissue-specific promoters, whereby skin-specific promoters such as the human K10 promoter (Bail leul et al., 1990. Cell 62: 697-708), the human K14 promoter (Vassar et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1563-67) or the bovine cytokeratin IV Promoter (Fuchs et al., 1988; The biology of wool and hair (Ed .: G. E. Rogers,  et al.), pp. 287-309. Chapman and Hall, London / New York) are particularly preferable gen.

More examples of controllable elements that are tissue-specific expression in Enabling eukaryotes are promoters or activator sequences from promo tors or enhancers of genes that code for proteins that are only found in certain cell types are expressed.

Examples of controllable elements, the cell cycle-specific expression in Euka enable rytones, are promoters of the following genes: cdc25A, cdc25B, cdc25C, Cyclin A, Cyclin E, cdc2, E2F-1 to E2F-5, B-myb or DHFR (Zwicker J. and Müller R. (1997) Trends Genet. 13, 3-6). The use of cell cycle regulation lated promoters is particularly preferred in cases where the expression of Proliferate polypeptides or nucleic acids used according to the invention de cells should be limited.

An example of a regulatable element that the keratinocyte-specific Ex The FiRE element (Jaakkola et al., 2000, Gen. Ther., 7: 1640-1647). The FiRE element is AP-1 driven, by FGF- inducible so-called response element of the Syndecan-1 gene (Jaakkola et al., 1998, FASEB J., 12: 959-9).

Examples of regulatable elements that are metabolically specific in expression Allowing eukaryotes are promoters caused by hypoxia, by glucose deficiency, be regulated by phosphate concentration or by heat shock.

An example of an adjustable element that is skin-specific expression the FiRE element is made possible.  

To introduce the nucleic acids described above and thus expression of the polypeptide in a eu or prokaryotic cell The nucleic acid can enable transfection, transformation or infection acid as a plasmid, as part of a viral or non-viral vector. As Viral vectors are particularly suitable here: baculoviruses, vaccinia viruses, ade noviruses, adeno-associated viruses and herpes viruses. As non-viral vectors are particularly: Virosomes, liposomes, cationic lipids, or po ly-lysine conjugated DNA.

Examples of gene therapy-effective vectors are virus vectors, at for example adenovirus vectors or retroviral vectors (Lindemann et al., 1997, Mol. Med. 3: 466-76; Springer et al., 1998, Mol. Cell. 2: 549-58). Eukaryo Expression vectors in isolation are suitable for gene therapy cal application, since bare DNA enters skin cells when applied topically (Hengge et al., 1996, J. Clin. Invest. 97: 2911-6; Yu et al., 1999, J. Invest. Dermatol. 112: 370-5).

Vectors with gene therapy effects can also be obtained by the nucleic acid described above complexed with liposomes, since there with a very high transfection efficiency, especially of skin cells (Alexander and Akhurst, 1995, Hum. Mol. Genet. 4: 2279-85). at In lipofection, small unilamellar vesicles become cationic lipids by ultrasound treatment of the liposome suspension. The DNA will bound ionically on the surface of the liposomes, in such a way Ratio that a positive net charge remains and the plasmid DNA too 100% is complexed by the liposomes. In addition to those of Felgner et al. (1987, supra) used lipid mixtures DOTMA (1,2-dioleyloxpropyl-3- trimethylammonium bromide) and DPOE (dioleoylphosphatidylethanolamine) which has since synthesized numerous new lipid formulations and tailored them to their needs Efficiency in transfection of different cell lines tested (Behr et al. (1989),  Proc. Natl. Acad. Sci. USA 86, 6982-6986; Gao and Huang (1991), Biochim. Bi ophys. Acta 1189, 195-203; Felgner et al. (1994) J. Biol. Chem. 269, 2550-2561). Examples of the new lipid formulations are DOTAP N- [1- (2,3- Dioleoyloxy) propyl] -N, N, N-trimethylammonium ethyl sulfate or DOGS (TRANSFECTAM; dioctadecylamidoglycyl spermine). As very well suited for that Transfection of keratinocytes in vitro and in vivo have also become the cationi lipids cytofectin GS 2888 (US 5,777,153; Lewis et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 3176-3181). Excipients that facilitate the transfer of Increase nucleic acids in the cell, for example proteins or peptides, that are bound to DNA or synthetic peptide DNA molecules that the Enable transport of the nucleic acid into the nucleus of the cell (Schwartz et al. (1999) Gene Therapy 6, 282; Brandén et al. (1999) Nature Biotech. 17, 784). Excipients also include molecules that inhibit the release of nucleic acids enable the cytoplasm of the cell (Planck et al. (1994) J. Biol. Chem. 269, 12918; Kichler et al. (1997) Bioconj. Chem. 8, 213) or, for example, Liposo men (Uhlmann and Peymann (1990) supra). Another particularly suitable one The form of gene therapy vectors can be obtained by applies the nucleic acid described above to gold particles and this with the help of the so-called "gene gun" in tissue, preferably in the skin, or Cells shoots (Wang et al., 1999, J. Invest. Dermatol., 112: 775-81, Tuting et al., 1998, J. Invest. Dermatol. 111: 183-8).

A further embodiment of a gene therapy-effective inventive According to the vector that can be used, the introduction of "bare" expresses ion vectors into a biocompatible matrix, for example a collagen matrix, produce. This matrix can be placed in wounds around which the wound derfenden cells with the expression vector to transfect and the fiction to express appropriate polypeptides in the cells (Goldstein and Banadio, US 5,962,427).  

For gene therapy use of the previously described nuclein It is also advantageous if the part of the nucleic acid that is responsible for the poly peptide encoding, including one or more non-coding sequences Intron sequences, preferably between the promoter and the start codon of the poly peptide, and / or a polyA sequence, especially the naturally occurring one polyA sequence or an SV40 virus polyA sequence, especially at the 3 'end of the Gens contains, since this can stabilize the mRNA (Palmiter et al., 1991, Proc. Natl. Acad. Sci. USA 88: 478-482; Jackson, 1993, Cell 74: 9-14).

Knockout gene constructs are known to the person skilled in the art, for example, from the US patents 5,625,122; US 5,698,765; US 5,583,278 and US 5,750,825 known.

Another preferred embodiment of the present invention is Ver using a cell, preferably an autologous or a heterologous Cell, in particular a skin cell, which can be used with an agent according to the invention Vector or knock-out gene construct for diagnosis and / or prevention and / or Treatment of diseases of skin cells and / or wound healing and / or their pathological disorders and for the identification of pharmacolo gisch active substances is transformed. Cells can be both prokaryotic as well as eukaryotic cells, examples of prokaryotic cells are E. coli and for eukaryotic cells Saccharomyces cerevisiae or insect cells. Examples of skin cells are keratinocytes, fibroblasts and endothelial cells.

A particularly preferred transformed cell that can be used according to the invention is a transgenic embryonic non-human stem cell, which is characterized by is that they have at least one knock-out usable according to the invention Gene construct and / or at least one expressi that can be used according to the invention on cassette, as described above. Transformation process  of cells and / or stem cells are well known to those skilled in the art and include for example electroporation or microinjection.

Transgenic non-human mammals whose genome contains at least one invention according to usable knock-out gene construct and / or at least one invented Expression cassette usable according to the invention, as described above may contain for the diagnosis and / or prevention and / or treatment of Diseases of skin cells and / or wound healing and / or their sick adhere to disorders or to identify pharmacologically active substances zen, can be used. Transgenic animals that are one of those described above Expression cassettes contain, depending on the promo used tors generally a tissue-specific increased expression of the nucleic acids and / or polypeptides and can be used to analyze wound healing disorders use. For example, an Activin A transgenic mouse has a verb Enhances wound healing (Munz et al., 1999, EMBO J. 18: 5205-15) during one transgenic mouse with dominant negative KGF receptor a delayed wound has healing (Werner et al., 1994, Science 266: 819-22). In addition, NEN previously described transgenic animals with accelerated wound healing be equipped.

Process for the production of transgenic animals, in particular transgenic Mice are also known to those skilled in the art from DE 196 25 049 and the US 4,736,866; US 5,625,122; US 5,698,765; US 5,583,278 and US 5,750,825 knows and include transgenic animals, for example, via direct injection of Expression vectors (see above) in embryos or spermatocytes or via the Transfection of expression vectors into embryonic stem cells can (Polites and Pinkert: DNA Microinjection and Transgenic Animal Production, pages 15 to 68 in Pinkert, 1994: Transgenic Animal Technology: A Laboratory Handbook, Academic Press, London, UK; Houdebine, 1997, Harwood Academic Publishers, Amsterdam, The Netherlands; Doetschman: Gene Transfer  in Embryonic Stern Cells, pages 115 to 146 in Pinkert, 1994, supra; Wood: retro virus-mediated gene transfer, pages 147 to 176 in Pinkert, 1994, supra; Mo nastersky: Gene Transfer Technology: Alternative Techniques and Applications, Pages 177 to 220 in Pinkert, 1994, supra).

Are the previously described nucleic acids usable according to the invention acids in so-called "targeting" vectors or "knock-out" gene constructs in tegriert (Pinkert, 1994, supra) after transfection of embryonic Stem cells and homologous recombination, for example knock-out mice Generated expresses generally reduced as heterozygous mice sion of nucleic acid show, while homozygous mice no expression of Have more nucleic acid. The animals produced in this way can also be analyzed use of wound healing disorders. For example, the eNOS (Lee et al., 1999, Am. J. Physiol. 277: H1600-H1608), Nf-1 (Atit et al., 1999, J. Invest. Dermatol. 112: 835-42) and Osteopontin (Liaw et al., 1998, J. Clin. Invest. 101: 967-71) knock-out mice impaired wound healing. Here too is a tissue-specific reduction in expression relevant to wound healing Genes, for example in skin-specific cells using the Cre-loxP Systems (stat3 knock-out, Sano et al., EMBO J. 1999 18: 4657-68), especially to prefer. Transgenic and knock-out cells or animals generated in this way can be also for screening (screening) and for the identification of pharmaco use logically active substances or gene therapy-active vectors.

2'-5'-Oligoadenylate synthetase and RNAse L Po which can be used according to the invention lypeptides Polypeptides can be prepared by generally known recombinant methods be produced. Furthermore, poly peptides isolated from an organism or from tissue or cells and invented be used properly. So it is possible, for example, fiction Moderately usable polypeptides from mammalian tissue, for example from skin or from body fluids, for example blood, serum, saliva, synovial fluid  or clean up wound fluid. Furthermore, according to the invention usable cells expressing polypeptides are produced then used to isolate polypeptides which can be used according to the invention (summarized in Player and Torrence, 998, Pharmacol. Ther., 78: 55-113). For example, expression vectors containing the inventions Nucleic acids which can be used according to the invention in skin cells, for example HaCaT Cells are transformed. The expression can be constitutive or, for example be inducible. It is preferably interferon-treated cells or tissue.

Furthermore, 2'-5'-oligoadenylate synthetase which can be used according to the invention and RNAse L polypeptides can be produced recombinantly. For example RNAse L prepared using the baculovirus system (Dong et al., 1994, J. Biol. Chem., 269: 14153-14158). 2'-5'-oligoadenylate synthetase proteins can also be produced recombinantly (e.g. Reboulliat and Hovanes sian, supra).

The 2'-5'-oligoadenylate synthetase or RNAse L polypeptide is used, for example by expression of the above-described nucleic acids in a suitable Neten expression system, as already mentioned above, according to the person skilled in the art made my known methods. For example, the cells are suitable E. coli strains DHS, HB101 or BL21, the yeast strain Saccharomyces cerevi siae, the insect cell line Lepidopteran, e.g. B. from Spodoptera frugiperda, or the animal cells COS, Vero, 293, HaCaT, and HeLa, all commonly available are.

Another embodiment relates to the use of the invention 2'-5'-oligoadenylate synthetase and / or RNAse L polypeptides, the poly peptides can be used in the form of a fusion protein. According to the invention Reversible fusion proteins can be made, for example, by inventing  nucleic acids of a suitable cell that can be used in accordance with the invention the.

The fusion proteins themselves already function as a 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide of the invention functional only after the fusion component has been split off. Above all, this includes Fu ion proteins with a proportion of about 1-300, preferably about 1-200, in particular about 1-100, especially about 1-50 foreign amino acids. Examples of such pep tidsequenzen are prokaryotic peptide sequences z. B. from galactosidase can be derived from E. coli. Viral peptide sequences can also be used zen, such as used by the bacteriophage M13, so Fusi to generate onsproteins for the "phage display" process known to the person skilled in the art gene.

Further preferred examples of peptide sequences for use according to the invention bare fusion proteins are peptides that facilitate the detection of the fusion proteins tern, these include, for example, "green fluorescent protein" (WO 95/07463) or functional variants of it.

For the purification of the 2'-5'-oligoadenylate synthetase and RNAse L polypeptides described above, a further / further polypeptide ("tag") can be added. Protein tags according to the invention allow, for example, high affinity absorption onto a matrix, stringent washing with suitable buffers without eluting the complex to any appreciable extent, and then targeted elution of the absorbed complex. Examples of the protein tags known to the person skilled in the art are a (His) ₆ tag, a Myc tag, a FLAG tag, a hemagglutinin tag, glutathione transferase (GST) tag, intein with an affinity-chitin binding -tag or Maltose-binding protein (MBP) -tag. These protein tags can be located at the N, C, and / or internally.

Another embodiment of the invention relates to the use of an anti body or antibody fragments, preferably a polyclonal or mo noclonal antibody or antibody fragments for analysis, diagnosis, pre prevention and / or treatment of diseases of skin cells, wounds tion and / or their pathological disorders and its use for identification Ornamentation of pharmacologically active substances, characterized in that an antibody-producing organism with an inventive use baren 2'-5'-oligoadenylate synthetase or RNAse L polypeptide is immunized. Polyclonal and monoclonal antibodies against usable according to the invention 2'-5'-Oligoadenylate synthetase polypeptides are known (Hovanessian et al., 1987, supra; Chebath et al. 1987, J. Biol. Chem., 262: 3852-3857; Marie et al., 1989, Biochem. Biophys. Res. Commun., 160: 580-587).

For example, the local injection of monoclonal antibodies against TGF beta 1 in animal models improve wound healing (Ernst et al., 1996, Gut 39: 172-5).

The procedure for making an antibody or antibody fragment preferably a polyclonal or monoclonal antibody occurs after Methods generally known to those skilled in the art by immunizing a mammal, for example a rabbit, with the 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide or functional variants thereof, preferably with parts thereof, with a length of at least 6 amino acids, preferably with at least 8 amino acids in length, in particular with at least 12 amino acids in length, optionally in the presence of e.g. B. Freund's Adju vant and / or aluminum hydroxide gels (see e.g. Diamond, B.A. et al. (1981) The New England Journal of Medicine, 1344-1349). The in the animal due to one Polyclonal antibodies resulting from immunological reaction can be then isolate easily from the blood using well known methods and Z. B. clean over column chromatography. Monoclonal antibodies can  For example, using the well-known Winter & Milstein method (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299). As an alternative to The classic antibodies can be based on lipocalin, for example called "Anticaline" can be used (Beste et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 1898-1903). Lipoca's natural ligand binding sites line, such as B. the retinol-binding protein or the bilin-binding protein can for example by a "combinatorial protein design" approach in one Modified so that they are selected haptens, such as the bind polypeptides which can be used according to the invention (Skerra, 2000, Biochim. Bi ophys. Acta 1482: 337-50). Other well-known "scaffolds" as alternatives for An Antibodies for molecular recognition are known (Skerra, J. Mol. Recognit., 2000, 13: 167-187).

The antibody or the antibody fragment which can be used according to the invention are directed against a 2'-5'-oligoadenylate synthetase or RNAse L polypeptide according to the invention and react specifically with the 2'-5'-oligoadenylate synthetase or RNAse L polypeptides according to the invention, the above-mentioned parts of the polypeptide either themselves are immunogenic or by coupling to suitable carriers, such as. B. bovine serum albumin, immunogenic or can be increased in their immunogenicity. This antibody which can be used according to the invention is either polyclonal or monoclonal, a monoclonal antibody is preferred. According to the present invention, the term antibody or antibody fragment is also understood to mean genetically produced and possibly modified antibodies or antigen-binding parts thereof, such as, for. B. chimeric antibodies, humanized antibodies, multifunctional antibodies by, bi- or oligo-specific antibodies, single-stranded antibodies, F (ab) - or F (ab) ₂ fragments (see, for example, EP-B1-0 368 684, US 4,816,567 , US 4,816,397, WO 88/01649, WO 93/06213, WO 98/24884).

The present invention also relates to the use of at least one invention 2'-5'-oligoadenylate synthetase and / or RNAse L which can be used according to the invention Polypeptide or a functional variant thereof or this encoding nuc linseic acid or a variant thereof, or a one which can be used according to the invention res 2'-5'-oligoadenylate synthetase and / or RNAse L polypeptide or a funk tional variant thereof or a nucleic acid encoding this or a Variant thereof expressing cell, or one against an invention usable 2'-5'-oligoadenylate synthetase and / or RNAse L polypeptide directed antibody or an antibody fragment, optionally combined or together with suitable additives and auxiliary substances, for the production of a Medicament for the prevention and / or treatment of diseases of the skin cells, wound healing and / or their pathological disorders.

The invention further relates to the use of a medicament for prevention tion and / or treatment of diseases of skin cells, wound healing and / or their pathological disorders, at least one according to the invention usable 2'-5'-oligoadenylate synthetase and / or RNAse L polypeptide or a functional variant thereof or this coding nucleic acid, or one a 2'-5'-oligoadenylate synthetase which can be used according to the invention and / or RNAse L polypeptide or a functional variant thereof or one of these nucleic acid or a cell expressing a variant thereof, or one against a 2'-5'-oligoadenylate synthetase which can be used according to the invention and / or RNAse L polypeptide or a functional variant thereof directed antibody pers or an antibody fragment, optionally combined or together with suitable additives and auxiliaries.

Therapy of diseases of skin cells and / or wound healing and / or their pathological disorders, can in a conventional manner, e.g. B. by Bandages, plasters, compresses or gels are made using the inventive Contain medicines. So it is possible to find the suitable additives or auxiliaries,  such as B. physiological saline, demineralized water, stabilizer ren, proteinase inhibitors, gel formulations, such as. B. white petroleum jelly, thinly flowing Paraffin and / or yellow wax, etc., containing pharmaceuticals topically and to be administered locally to immediately and immediately influence wound healing sen. The administration of the medicament according to the invention can also be used optionally in the form of liposome complexes or gold particle complexes also topically and locally in the area of the wound. Furthermore, the Treatment using a transdermal therapeutic system (TTS), which is a time-controlled delivery of the medicament which can be used according to the invention medium enables. Treatment by means of those which can be used according to the invention Medicines can also be taken via oral dosage forms, such as. B. tablets or capsules, over the mucous membranes, for example the nose or the oral cavity, or in the form of disposers implanted under the skin. TTS are for example from EP 0 944 398, EP 0 916 336, EP 0 889 723 or EP 0 852 493 known.

For gene therapy use in humans is primarily a drug means suitable that the nucleic acid described in naked form or in the form one of the gene therapy vectors described above or in with Contains liposomes or gold particles of complex form. The pharmaceutical The carrier is, for example, a physiological buffer solution, preferably with egg pH of about 6.0-8.0, preferably about 6.8-7.8. In particular from about 7.4 and / or an osmolarity of approx. 200-400 milliosmol / liter, preferably of approx. 290-310 milliosmol / liter. In addition, the pharmaceutical carrier can be used Nete stabilizers such. B. nuclease inhibitors, preferably complexing agents such as EDTA and / or other auxiliaries known to those skilled in the art.

The administration of the nucleic acid described optionally in the form of Virus vectors described in more detail above or as liposome complexes or Gold particle complex usually takes place topically and locally in the area of  Wound. It is also possible to add or add the polypeptide itself Excipients such as B. physiological saline, demineralized water, Stabilizers, proteinase inhibitors, gel formulations, such as. B. white Vaseli ne, low-viscosity paraffin and / or yellow wax, etc., to administer the Influence wound healing immediately and immediately.

Examples of diseases of the skin cells in the sense of the present invention are psoriasis, eczema, especially atopic eczema, acne, urticaria, pig Skin disorders, especially vitiligo, "senile skin" and disorders of the skin Hair growth or metabolism.

Wound healing in the sense of the present invention is the healing process to understand a mechanical injury to the skin, such as B. cuts, Grazes or rubbing of the skin, for example, by persistent loading burden, for example pressure ulcers or necrotic processes, for example Necrobiosis lipoidica.

Examples of impaired wound healing in the sense of the present invention are Wounds of diabetic patients and alcoholics, infected with organisms or viruses graced wounds, ischemic wounds, wounds from patients with arterial Occlusive diseases or venous insufficiency, and scars, preferably over shooting scars, especially keloids. Particularly preferred to heal poorly en Wounds are diabetic, neuropathic, venous and arterial ulcers, especially special venous ulcers.

The present invention further relates to the use of at least one 2'-5'-oligoadenylate synthetase or RNAse L which can be used according to the invention Polypeptide or a functional variant thereof or this encoding nuc linseic acid or a variant thereof, or a usable according to the invention 2'-5'-oligoadenylate synthetase or RNAse L polypeptide or a functional one  Variant thereof or a nucleic acid coding therefor or variant thereof expressing cell, or one against a 2'-5'- usable according to the invention Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof directed antibody or an antibody fragment if combined or together with suitable additives and auxiliaries, for the manufacture provision of a diagnostic agent for the diagnosis of diseases of skin cells and / or in wound healing and / or its pathological disorders.

For example, according to the present invention, a diagnostic agent based on the polymerase chain reaction (examples 2 to 6 PCR diagnostics, e.g. according to EP 0 200 362) or an RNase protection assay (see e.g. B. Sambrook et al., Supra chapter 7, pages 7.71-7.78; Werner et al., 1992, Growth Factors and Receptors: A Practical Approach 175-197; Werner, 1998, Proc. Natl. Acad. Sci. USA 89: 6896-6900). These tests are based on the specific hybridization of a nucleic acid with its complementary counter strand, usually the corresponding mRNA or its cDNA. The nucleic acids that can be used according to the invention can also be modified, such as. B. disclosed in EP 0 063 879. Such a DNA fragment is preferably prepared using suitable reagents, e.g. B. radioactively labeled with α-P ³² -dCTP or non-radioactively with biotin or digoxigenin, according to generally known methods and with isolated RNA, which are preferably attached to suitable membranes from z. B. was bound nitrocellulose or nylon, incubated. With the same amount of RNA examined from each tissue sample, the amount of mRNA that was specifically labeled by the probe can thus be determined. Alternatively, the amount of mRNA can also be determined directly in tissue sections using in situ hybridization (see, for example, Werner et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6896-900).

The present invention further relates to the use of a diagnostic cumulative for the diagnosis of diseases of skin cells and / or for wounds  healing and / or their pathological disorders, at least one invention usable according to 2'-5'-oligoadenylate synthetase or RNAse L polypeptide or a functional variant thereof or nucleic acid encoding it or a variant thereof, or a 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof or a nucleic acid coding therefor or a variant thereof expressing cell, or a 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof directed antibody or an antibody fragment, if appropriate combined or together with suitable additives and auxiliary substances, for diagnosis diseases of skin cells and / or wound healing and / or their pathological disorders is used.

With the help of the diagnostic agent which can be used according to the invention, therefore, also in the specific expression level of the respective gene in vitro in a tissue sample can be measured, for example, a wound healing disorder or skin diagnose diseases safely (Examples 2, 5, 6). In particular Such a method is suitable for the early prognosis of faults.

A preferred diagnostic agent according to the invention contains one according to the invention usable 2'-5'-oligoadenylate synthetase or RNAse L polypeptide or the immunogenic parts thereof described in more detail above. The polypeptide or Parts thereof, which are preferably connected to a solid phase, e.g. B. from nitrocellulose or Nylon, for example, can be bound with the body to be examined liquid, e.g. B. wound secretion, in vitro in contact, so for example to be able to react with autoimmune antibodies. The antibody The peptide complex can then be identified, for example, using labeled anti-human IgG or anti-human IgM antibodies are detected. At the mark for example, it is an enzyme, such as peroxidase, that has a color craze  tion catalyzed. The presence and amount of autoimmunanti present body can thus be easily and quickly detected via the color reaction.

Another diagnostic agent that can be used according to the invention, the subject of the present invention contains the antibody which can be used according to the invention by yourself. With the help of these antibodies, for example, a tissue sample easily and quickly examined whether the polypeptide in question is present in an increased amount, thereby indicating a possible disease, especially skin diseases and wound healing disorders receive. In this case, the antibodies according to the invention are, for example labeled with an enzyme as described above. The specific anti body-peptide complex can be easily and just as quickly via an enzy matic color reaction can be detected.

Another diagnostic agent which can be used according to the invention comprises a probe, preferably a DNA probe, and / or primer. This opens up another one Possibility of the described nucleic acids, for example by isolation from a suitable gene bank, for example from a wound-specific gene bank using a suitable probe (see, e.g., J. Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Chapter 8 pages 8.1 to 8.81, Chapter 9 pages 9.47 to 9.58 and Chapter 10 page 10.1 to 10.67).

For example, DNA or RNA fragments with a length are suitable as a probe ge of about 100-1000 nucleotides, preferably with a length of about 200-500 Nucleotides, in particular with a length of about 300-400 nucleotides Sequence from the 2'-5'-oligoadenylate synthetase or RNAse L polypeptides according to SEQ ID No. 1 to SEQ ID No. 4 and SEQ ID No. 9 to SEQ ID No. 12 of the sequence listing and / or using the cDNA sequences shown in Table 1 specified database entries or using the sequence listing according to a  SEQ ID No. 5 to SEQ ID No. 8 and SEQ ID No. 13 to SEQ ID No. 14 can be.

Alternatively, oligonucleotides can be derived from the derived nucleic acid sequences de be synthesized, which acts as a primer for a polymerase chain reaction suitable. The nucleic acid or parts described above can be used with these this from cDNA, for example wound-specific cDNA, amplified and iso be liert (Examples 2 to 6). Suitable primers are, for example, DNA Fragments with a length of about 10 to 100 nucleotides, preferably with 15 to 50 nucleotides in length, in particular 20 in length up to 30 nucleotides whose sequence from the 2'-5'-oligoadenylate synthetase or RNAse L polypeptides according to SEQ ID No. 1 to SEQ ID No. 4 and SEQ ID No. 9 to SEQ ID No. 12 of the sequence listing and / or using the cDNA Se sequences of the database entries specified in Table 1 or based on Se sequence protocol according to SEQ ID No. 5 to SEQ ID No. 8 and SEQ ID No. 13 up to SEQ ID No. 14.

The invention further relates to the use of at least one inven tion usable 2'-5'-oligoadenylate synthetase or RNAse L polypeptide or a functional variant thereof or this encoding nucleic acid or a variant thereof, or a 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof or a nucleic acid coding therefor or a variant thereof expressing cell, or one against a 2'-5'- usable according to the invention Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof directed antibody or an antibody fragment if combined or together with suitable additives and auxiliary substances, for I diagnosis of pharmacologically active substances related to Diseases of skin cells and / or wound healing and / or their sick adhere to disturbances.  

The present invention further relates to the use of at least one 2'-5'-oligoadenylate synthetase or RNAse L which can be used according to the invention Polypeptide or a functional variant thereof or this encoding nuc linseic acid or a variant thereof, or a usable according to the invention 2'-5'-oligoadenylate synthetase or RNAse L polypeptide or a functional one Variant thereof or a nucleic acid coding therefor or variant thereof expressing cell, or one against a 2'-5'- usable according to the invention Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof directed antibody or an antibody fragment if combined or together with suitable additives and auxiliaries, for the manufacture provision of a test for the detection of pharmacologically active substances in Relation to skin disorders and / or wound-related healing, in particular wound healing disorders.

Under the term "pharmacologically active substances" in the sense of vorlie Invention are all those molecules, compounds and / or Zu to understand compositions and mixtures of substances that with the preceding be written nucleic acids, nucleic acid analogs, polypeptides, antibodies or Antibody fragment, if necessary together with suitable additives and auxiliaries substances can interact under suitable conditions. Moegli Interactors are simple chemical organic or inorganic molecules or compounds, but can also nucleic acids, peptides, proteins or Complexes of these include. The interactors can because of their interaction the function (s) of the nucleic acids, polypeptides or antibodies in vivo or influence in vitro or only to the previously described nuc bind or with linseed acids, polypeptides, antibodies or antibody fragments other interactions covalently or non-covalently hen.  

The present invention further relates to the use of an on test discovery of pharmacologically active substances in connection with crane skin cell and / or wound healing and / or pathological Disorders, with at least one 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof or nucleic acid coding therefor or a variant thereof, or a 2'-5'-oligoadenylate synthetase which can be used according to the invention or RNAse L polypeptide or a functional variant thereof or one of these coding nucleic acid or variant thereof expressing cell, or a ge gene a 2'-5'-oligoadenylate synthetase usable according to the invention or RNAse L polypeptide or a functional variant thereof directed antibody or an antibody fragment, optionally combined or together with ge suitable additives and auxiliaries.

Another embodiment of the invention relates to the use at least of a polypeptide which can be used according to the invention, or a coding nucleus linseic acid, or a polypeptide or one which can be used according to the invention this encoding nucleic acid-expressing cell, or one against an invented A polypeptide-directed antibody or an anti usable according to the invention body fragments, optionally combined or together with suitable supplements Substances and auxiliaries, for the detection of pharmacologically active substances in connection with skin diseases and / or in connection with wound healing, in particular wound healing disorders, the pharmacologically active ven substances the activity of at least one usable according to the invention usable 2'-5'-oligoadenylate synthetase or RNAse L polypeptide influence. For this purpose, a pharmacologically active substance with the Invention according to usable polypeptide and the change the activity can be determined, or the activity with that of untreated Polypeptide are compared.  

Suitable assays for determining the activity of the ba Ren 2'-5'-oligoadenylate synthetase and RNAse L polypeptides are known to the person skilled in the art known and are with Reboulliat and Hovanessian (1999, supra) and Player and Torrence (1998, supra).

To determine the activity of a 2'-5'-oligoadenylate synthetase 2-5A Synthetase activity using an enzyme assay with subsequent determination of the end product. be determined. This can be done using radioactive labeled ATP. The subsequent analysis of the 2-5A products can then by means of HPLC, column chromatography, thin layer chromatography, E electrophoresis or DEAE paper binding (Player and Torrence, 1999, supra; P. 69). Furthermore, 2-5A can be detected using antibodies conditions that recognize 2-5A end products. A coupled assay can also be used of both a 2'-5'-oligoadenylate synthetase polypeptide and a Contains RNAse L polypeptide. The activity of one by pharmacologically active Substance-modulated 2'-5'-oligoadenylate synthetase polypeptide can then can be detected by measuring the RNAse activity. Another indirect The test consists of inhibiting protein synthesis in a cell-free To measure the system by degradation of the RNA (Player and Torrence, 1999, sup ra; P. 69). This can also be freed up by converting ATP to 2-5A set pyrophosphate can be determined by a spectrophotometric assay.

To determine the activity of the RNAse L, either the 2-5A binding o which determine the RNAse activity. The 2-5A bond can, for example using radio affinity labeling as in Floyd-Smith et al. (1982, J. Biol. Chem., 257: 8584-8587) or Nolan-Sorden et al. (1990, Anal. Biochem., 184: 298-304) or by competitive binding with radioactive sub such as Johnston and Torrence (1984; In: Interferon: Me chanism of Production and Action, pages 189-298, Elsevier) the. A nuclease assay can also be carried out. Suitable nuclea  These assays consist of gel electrophoretic analysis more specific or un specific RNAse cleavage products (Wreschner et al., 1981, Nucleic Acid Res., 9: 1571-1581; Williams et al., 1981, Methods Enzymol., 79: 199-208), Degrada tion of radiolabeled poly (U) by RNAse L on 2-5A cellulose (Sil verman, 1985, anal. Biochem., 144: 450-460) and the determination of acid precipitable Radioactivity (Biglioni et al., 1981, J. Biol. Chem., 256: 3253-3257).

In one embodiment of the present invention is used for identification of pharmacologically active substances related to diseases of skin cells and / or in wound healing and / or their pathological disturbances at least one 2'-5'- Oligoadenylate synthetase or RNAse L polypeptide or a functional Vari ante thereof or a nucleic acid coding therefor or a variant thereof expressing cell is used.

A suitable system can be, for example, through the stable transformation of epidermal or dermal cells with expression vectors that are selectable Contain marker genes and contain the described nucleic acids. At the This method is the expression of the nucleic acids described in the cell len changed so that it corresponds to the pathologically disturbed expression in vivo. Anti-sense oligonucleotides that contain the described nucleic acid, can be used for this purpose. Of particular advantage for this Systems is therefore the expression behavior of the genes in disturbed regenerati processes known as disclosed in this application. Often it can pathological behavior of the cells can be mimicked in vitro and it can Substances are sought that restore the normal behavior of the cells len and who have a therapeutic potential.

For these test systems that can be used according to the invention, z. B. HaCaT- Cells that are commonly available and the expression vector pCMV4 (other  son et al., 1989, J. Biol. Chem. 264: 8222-9). The nuk described above Linseed acid can be used in both sense and anti-sense orientation Expression vectors are integrated so that the functional concentration mRNA of the corresponding genes in the cells either increased, or by Hybridization with the antisense RNA is decreased. After the transformation and selection of stable transformants generally show the cells in culture a changed proliferation, migration, and / or differentiation behavior compared to control cells. This behavior in vitro is common with the radio tion of the corresponding genes in regenerative processes in the organism correct (Yu et al., 1997, Arch. Dermatol. Res. 289: 352-9; Mils er al., 1997, Oncoge ne 14: 15555-61; Charvat et al., 1998, Exp Dermatol 7: 184-90; Werner, 1998, Cytokine Growth Factor Rev. 9: 153-65; Mythily et al., 1999, J. Gen. Virol. 80: 1707-13) and can be carried out with simple and quick tests demonstrate, so that based on it test systems for pharmacologically active Can build up substances. This is how the proliferation behavior of cells can be very quickly through z. B. the incorporation of labeled nucleotides in the DNA of the Cells (see, e.g., Savino and Dardenne, 1985, J. Immunol. Methods 85: 221-6; Perros and Weightman, 1991, Cell Prolif. 24: 517-23; de Fries and Mitsuhashi, 1995, J. Clin. Lab. Anal. 9: 89-95), by staining the cells with specific Dyes (Schulz et al., 1994, J. Immunol. Methods 167: 1-13) or via immu biological methods (Frahm et al., 1998, J. Immunol. Methods 211: 43-50) prove. The migration can be done easily with the "Migration Index" test (Charvat et al., Supra) and comparable test systems (Benestad et al., 1987, Cell Tissue Kinet. 20: 109-19, Jung et al., 1993, J. Immunol. Methods 160: 73-9) prove. As differentiation markers such. B. Keratin 6, 10 and 14 and Loricrin and Involucrin (Rosenthal et al., 1992, J. Invest. Dermatol. 98: 343- 50), the expression of which, for. B. easily available via commonly available antibodies is pointing.  

Another suitable test system that can be used according to the invention is based on the Identification of interactions with the so-called "two-hybrid system" (Fields and Sternglanz, 1994, Trends in Genetics, 10, 286-292; Colas and Brent, 1998 TIBTECH, 16, 355-363). In this test, cells with expressions transformed vectors, the fusion proteins from the poly according to the invention peptide and a DNA binding domain of a transcription factor as in for example express Gal4 or LexA. The transformed cells contain also a reporter gene, the promoter of which corresponds to binding sites for the de DNA binding domain included. By transforming another ex pressure vector, which is a second fusion protein from a known or unbe known polypeptide with an activation domain, for example from Gal4 or Herpes simplex virus VP16, expressed, can express the reporter gene be greatly increased if the second fusion protein with the fiction interacts with the polypeptide. You can take advantage of this increase in expression, to identify new interactors, for example by going to the construct on the second fusion protein a cDNA library from regenerating Fabric. In addition, this test system can be used for grid examination of Exploit substances that interact between the invention Inhibit polypeptide and an interactor. Such substances reduce the Expression of the reporter gene in cells, the fusion proteins of the invention expression of the polypeptide and the interactor (Vidal and Endoh, 1999, Trends in Biotechnology, 17: 374-81). This way, new active ingredients can be identified quickly Ren, which are used for the treatment of disorders of regenerative processes can.

Another test to identify pharmacologically active substances has been passed in that a 2'-5'-oligoadenylate synthetase usable according to the invention or RNAse L polypeptide or a functional variant thereof or one of the coding nucleic acid or variant thereof expressing cell is brought into contact with pharmacologically active substances, the activity of  2'-5'-oligoadenylate synthetase or the RNAse L polypeptide is determined and is compared with the activity of untreated cells.

Furthermore, a test system can be based on the fact that according to the invention ren polypeptides or functional variants thereof or this encoding nuc linseed acids or variants thereof, or against buttocks which can be used according to the invention lypeptides or functional variants thereof directed antibodies or antibodies per fragments are bound to a solid phase and substances interact, For example, binding or changing the conformation can be tested. Geeig Systems such as affinity chromatography and fluorescence spectroscopy are known to the person skilled in the art.

The solid phase-bound polypeptides which can be used according to the invention or functional variants thereof or these encoding nucleic acids or a Variant thereof, or against a polypeptide or usable according to the invention a functional variant thereof directed antibody or an antibody question ment can also be part of an array. Process for the production of such Arrays are, for example, from US Pat. No. 5,744,305 using solid-phase chemistry and known photolabile protecting groups. These arrays can also be used with Substan zen or substance libraries are brought into contact and interact, for example binding or changing the conformation.

For example, a substance to be tested can have a detectable marker contain, for example, the substance can be radiolabelled, fluorescence marked or luminescence marked. Furthermore, substances can be added to Pro teine that allow indirect detection, for example via enzymatic catalysis using a peroxidase assay with a chromogenic Substrate or by binding a detectable antibody. Changes in Conformation of a polypeptide which can be used according to the invention by interaction  on with a test substance, for example, by changing the fluorescence of an endogenous tryptophan residue in the polypeptide can be detected.

Pharmacologically active substances of the poly usable according to the invention peptides can also be nucleic acids, which are via selection methods, as in for example SELEX (see Jayasena, 1999, Clin. Chem. 45: 1628-50; Klug and Famulok, 1994, M. Mol. Biol. Rep. 20: 97-107; Toole et al., 1996, US 5,582,981) be isolated. The SELEX process typically consists of a large Pool of different single-stranded RNA molecules through repeated ampli Specification and selection isolated those molecules that are attached to an invention bind usable polypeptide with high affinity (aptamers). Aptamers can also in their mirror image form, for example as an L-ribonucleotide, are synthesized and selected (Nolte et al., 1996, Nat. Biotechnol. 14: 1116-9; Klussmann et al., 1996, Nat. Biotechnol. 14: 1112-5). For isolated Men have the advantage that they are not from naturally occurring ribonucleases are broken down and therefore have greater stability.

Pharmacologically active substances of the poly usable according to the invention peptides can also be nucleic acid analogs of 2-5A. So far meh rere 2-5 derivatives identified as antagonists of the 2-5 effect (Lesiak and Tor rence, 1986, J. Med. Chem., 29: 1015-1022; Imai et al., 1982, J. Biol. Chem, 257: 12,739 to 12,745; Player and Torrence, 1998, supra, p. 80).

The identified using the test methods that can be used according to the invention pharmacologically active substances, optionally combined or together with suitable additives and auxiliaries, for the preparation of a Di agnostic or medicinal product for diagnosis, prevention and / or treatment diseases of skin cells, wound healing and / or their pathological Disturbances are used.  

Another embodiment of the invention relates to the use at least a polypeptide which can be used according to the invention, or a nuc which encodes it linseic acid, or a polypeptide or one which can be used according to the invention this encoding nucleic acid-expressing cell, or one against an invented A polypeptide-directed antibody or an anti usable according to the invention body fragments, optionally combined or together with suitable supplements Substances and auxiliaries, for the detection of pharmacologically active substances in connection with skin diseases and / or in connection with wound healing, in particular wound healing disorders, the pharmacologically active ven substances the expression of at least one usable according to the invention Affect nucleic acid.

Assays to identify pharmacological substances that cause expression of genes are generally known to the person skilled in the art (see, for example see Sivaraja et al., 2001, US 6,183,956).

For example, cells that express 2'-5'-oligoadenylate synthetase or RNAse L can for example HeLa cells, as a test system for analyzing gene expression in are cultivated in vitro, skin cells, in particular keratinocytes, Fibroblasts or endothelial cells are preferred. A possible test system represents the human keratinocyte cell line HaCaT, which he generally is stable.

Gene expression is analyzed, for example, at the mRNA level or the proteins. The amount of 2'-5'-oligoadenylate synthetase and / or RNAse L mRNA or protein after addition of one or more sub punch to the cell culture and measured with the appropriate amount in one Control culture compared. This is done, for example, with the help of a hybrid anti-sense probe with which the mRNA of  usable 2'-5'-oligoadenylate synthetase and / or RNAse L detected can be. The hybridization can be done, for example, by binding a specific antibody to the mRNA probe complex can be quantified (see Stuart and Frank, 1998, US 4,732,847). It is possible to do the analysis to carry out in the high throughput process and very many substances on their Suitability as a modulator of the expression of 2'-5'-oligoadenylate synthetase or Analyze RNAse L (Sivaraja et al., 2001, US 6,183,956). The analysis substances can be obtained from substance libraries (see e.g. DE 198 16 414, DE 196 19 373) can be taken, the several thousand, often very contain heterogeneous substances. Alternatively, the entire RNA or mRNA are isolated from cells, and then the absolute amount or the relative proportion of the mRNA of usable 2'-5'-oligoadenylate synthetase or RNAse L, for example, using quantitative RT-PCR (see EP 0 200 362; Wittwer et al., 1997, BioTechniques 22: 130-8; Morrison et al., 1998, BioTechni ques 24: 954-62) or the RNAse Protection Assay (see e.g. Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York, Chapter 7; EP 0 063 879) become. Another possibility is to analyze the amount of protein in the cell lily sat with antibodies that specifically 2'-5'-oligoadenylate synthetase or RNAse L recognize. Here, quantification can be done, for example, using an ELISA or a Western blot, which are generally known. To the specificity the substances on the expression of 2'-5'-oligoadenylate synthetase and / or Determining RNAse L can influence the influence of substances on the 2'-5'- Oligoadenylate synthetase and / or RNAse L expression with the influence on the Expression other genes, such as metabolic genes such as GAPDH. This can either be done in separate analyzes, or in parallel to the analysis of the 2'-5'-oligoadenylate synthetase and / or RNAse L. Heterodimers or its individual components.  

Another object of the invention relates to the use of at least one polypeptide or a functional variant which can be used according to the invention thereof or this coding nucleic acid or a variant thereof, or one against a polypeptide which can be used according to the invention or a functional variant ante thereof directed antibody or an antibody fragment if combined or together with suitable additives and auxiliaries, for the manufacture provision of an array fixed on a carrier material for analysis in compilation connection with diseases of skin cells and / or wound healing and / or their pathological disorders.

Methods for producing such arrays are, for example, from the US 5,744,305 by means of solid-phase chemistry and photolabile protective groups.

The present invention further relates to the use of such an Ar rays for analysis in connection with diseases of skin cells in which Wound healing and / or their pathological disorders, at least one invented usable polypeptide according to the invention or a functional variant thereof or this coding nucleic acid or a variant thereof, or one against one Polypeptide usable according to the invention or a functional variant thereof directional antibody or an antibody fragment, optionally combined or together with suitable additives and auxiliaries.

For analysis in connection with diseases of skin cells and / or of Wound healing and / or their pathological disorders can also, for example DNA chips and / or protein chips containing at least one nucleic acid, at least tens a polypeptide, and / or at least one antibody or antibody question ment as described above. For example, DNA chips are out the US 5,837,832 known.  

The invention will now be further described in the following using the tables and examples are illustrated without the invention being restricted thereto.

Description of the tables and sequences

Table 1: Tabular overview of 2'-5'-oligoadenylate synthetase and RNAse L polypeptide sequences which can be used according to the invention and their cDNAs and access numbers and / or SEQ ID numbers.
Table 2: Tabular listing of the changed expression of the 2'-5'-oligoadenylate synthetase gene in wounds of 10-week-old BALB / c mice and in wounds of young (4-week-old) and old (12-month) mice, as well as in mice with genetic diabetes.
Table 3: Analysis of the kinetics of the 2'-5'-oligoadenylates 26277 00070 552 001000280000000200012000285912616600040 0002010122206 00004 26158 Synthetase mRNA expression during mouse wound healing using "TaqMan analysis"
Table 4: Analysis of the kinetics of the 2'-5'-oligoadenylate synthetase mRNA expression during wound healing in humans by means of "TaqMan analysis"
Table 5: Analysis of the expression of the 2'-5'-oligoadenylate synthetase mRNA in human ulcer biopsies
Table 6: Analysis of the 2'-5'-oligoadenylate synthetase mRNA expression in single biopsies of lesional and non-lesional skin of psoriasis patients as well as in biopsies of intact skin of healthy volunteers.

SEQ ID No. 1 to SEQ ID No. 14 show 2'-5'- Oligoadenylate synthetase and RNAse L polypeptide or cDNA sequences Human or mouse.

SEQ ID No. 15 to SEQ ID No. 24 show DNA sequences of oligonucleotides, used for the experiments of the present invention.

Examples example 1 Enrichment of 2'-5'-oligoadenylate synthetase cDNA by means of subtractive hybridization and identification of the 2'-5'-oligoadenylate synthesis tase as a gene relevant to wound and skin disease

From intact skin and from wound tissue (wound on the back 1 day before Ge weaving by paper cutting) of BALB / c mice was carried out by Stan standard methods (Chomczynski and Sacchi, 1987, Anal. Biochem. 162: 156-159, Chomczynski and Mackey, 1995, Anal. Biochem. 225: 163-164) Total RNA isolated. To obtain tissue from mice with poorly healing wounds, BALB / c mice were injected with dexamethasone (injection of 0.5 mg dexamethasone in isotonic saline twice per kg body weight per day for 5 days). The RNAs were then reversed Transcriptase rewritten in cDNA. The cDNA synthesis was carried out with the "SMART PCR cDNA Synthesis Kit" from Clontech Laboratories GmbH, Heidelberg, according to the instructions in the relevant manual.

In order to identify those cDNAs with different frequencies in cDNA pools, subtractive hybridization (Diatchenko et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 6025-30). This he  followed with the "PCR-Select cDNA Subtraction Kit" from Clontech Labora tories GmbH, Heidelberg, according to the instructions in the relevant manual, whereby the separation of excess oligonucleotides after cDNA synthesis Agarose gel electrophoresis was performed. There were two for genes relevant to wounds enriched cDNA pools created, with a pool of cDNA fragments was in wound healing in comparison to bad healing wounds which are more strongly expressed ("well healing cDNA pool") and a pool was indicated enriches cDNA fragments compared to poorly healing wounds well-healing wounds are more strongly expressed ("poorly healing cDNA pool").

In order to identify those genes that are found in the cDNA Pools were included, the presence of the corresponding cDNAs was in the pools in the "Reverse Northern Blot" analyzed. Here are cDNA fragments fixed on membranes in the form of arrays of many different cDNAs, and hybridized with a complex mixture of radioactively labeled cDNA (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York, Chapter 9 page 9.47 to 9.58 and Chapter 10 page 10.38 to 10.50; Anderson and Young: Quantitative filter hybridization; in: Nucleic Acids Hybridization, A Practical Approach, 1985, Eds. Hames and Higgins, IRL Press Ltd .; Oxford, Chapter 4, pages 73 to 112). For example, commercially available membranes were used (mouse ATLAS Array, Clontech).

The subtracted were used to produce suitable hybridization probes cDNA pools treated with the restriction endonuclease RsaI and over agarose gel electrophoresis cleaned (Sambrook et al., supra, chapter 6, pages 6.1 to 6.35), for the cDNA synthesis and amplification primers (see manual "PCR-Select cDNA Subtraction Kit ", Clonetech). The cDNAs were then with the "random hexamer priming" method (Feinberg and Vogelstein, 1983, Anal. Bio chem. 132: 6-13) radioactively labeled to produce hybridization probes.  

The membrane was vorin for 30 min at 65 ° C in 25 ml hybridization solution cubed (25 mM sodium phosphate, pH = 7.5, 125 mM NaCl, 7% SDS). The Hybridization probe was denatured at 100 ° C for 10 min, then on ice cooled, approx. 100 CPM ("counts per minute") per ml to the hybridization solution given and the hybridization for 16 hours at 65 ° C in the hybridization oven carried out. The membrane was then rinsed twice with the Hybridi for 10 min wash solution without probe at 65 ° C. Then the Memb ran several times for 10 min each in washing solution (2.5 mM sodium phosphate, pH = 7.5, 12.5 mM NaCl, 0.7% SDS) washed at 65 ° C until poured into the Solution could no longer be detected. The radioactive sig nals were evaluated with a phosphoimager (BioRad, Quantitiy One®). Then those cDNAs were selected that were used with the different probes different signal intensities resulted. This resulted in the position of 2'-5'-oligoadenylate synthetase on the membrane has a significantly stronger signal intensity with the hybridization probe of the "poorly healing cDNA pool" in the Comparison to the "well healing cDNA pool".

Example 2 Verification of the Expression Pattern of the 2'-5'-Oligoadenylate Syn thetase (2-5 OAS) using "TaqMan Analysis"

A verification of the differential expression of the 2'-5'-oligoadenylate synthesis tase mRNA in wounds treated with dexamethasone as well as the studies Further wound healing states were carried out using "TaqMan Analysis" in the GeneAmp5700 from Applied Biosystems.

In order to obtain tissue from mice with poorly healing wounds, BALB / c mice before wounding with dexamethasone (0.5 mg injection Dexamethasone in isotonic saline twice a day per kg body weight for 5 days). To obtain tissue from young and old mice, became 1-day wounds from 4 week old and 12 month old BALB / c mice  sen used. To obtain wound tissue from mice with diabetes, 1 Daily wounds from 10 week old C57BL / Ks-db / db / Ola mice were used.

The RNA was isolated by homogenizing the biopsies in RNA clean buffer (AGS, Heidelberg), to which 1/100 volume fraction of 2-mercaptoethanol had been added, using a disperser. The RNA was then extracted by phenolizing twice using water-saturated acid phenol and in the presence of 1-bromo-3-chloropropane. An isopropanol and an ethanol precipitation were then carried out and the RNA was washed with 75% ethanol. A DNase I digestion of the RNA was then carried out. For this purpose 20 µg RNA (ad 50 µl with DEPC-treated water) with 5.7 µl transcription buffer (Roche), 1 µl RNase inhibitor (Roche; 40 U / µl) and 1 µl DNase I (Roche; 10 U / µl) incubated at 37 ° C for 20 min. Then again 1 ul DNase I was added and incubated at 37 ° C for a further 20 min. The RNA was then phenolized, ethanol precipitated and washed. All of the steps listed above were carried out with DEPC (diethyl pyrocarbonate) -treated solutions or liquids, provided that these did not contain any reactive amino groups. The cDNA was then produced from the extracted RNA. This was done in the presence of 1 × TaqMan RT buffer (Applied Biosystems), 5.5 mM MgCl ₂ (Perkin Elmer), 500 µM dNTPs (Perkin Elmer), 2.5 µM random hexamers (Perkin Elmer), 1, 25 U / µl MultiScribe Reverse Transcriptase (50 U / µl Perkin Elmer), 0.4 U / µl RNase Inhibitor (20 U / µl, Perkin Elmer), 20 µl RNA (50 ng / µl) and DEPC-treated water (ad 100 µl volume). After addition of the RNA and thorough mixing, the solution was distributed into 2 0.2 ml tubes (50 μl each) and the reverse transcription was carried out in a temperature cycler (10 min at 25 ° C; 30 min at 48 ° C and 5 min at 95 ° C). The following quantification of the cDNA was carried out by means of quantitative PCR using the SYBR Green PCR Master Mix (Perkin Elmer), whereby a triple determination (each with 2-5 OAS primers (mOAS primer 1 CCTTCCTCAA CAGATTCAGA AGGA (SEQ ID No. 17) and mOAS primer 2: TGATCAGACT TTGTCAGACA GAACCT (SEQ ID No. 18)) and GAPDH primers) was carried out. The stock solution for each triplet contained 37.5 µl 2 × SYBR Master Mix, 0.75 µl AmpErase UNG (1 U / µl) and 18.75 µl DEPC-treated water for a total volume of 57 µl. For each triple determination, 1.5 µl of forward and reverse primers were added to 57 µl stock solution in a previously optimized concentration ratio. 60 µl each of the stock solution / primer mixture was mixed with 15 µl cDNA solution (2 ng / µl) and distributed over 3 reaction vessels. In parallel, a stock solution with primers for the determination of GAPDH (GAPDH primer 1: ATCAACGGGA AGCCCATCA (SEQ ID No. 15) and GAPDH primer 2: GACATACTCA GCACCGGCCT (SEQ ID No. 16)) as a reference was prepared, with a further 15 μl the same cDNA solution mixed and distributed over 3 reaction vessels. In addition, in order to create a standard curve for GAPDH-PCR, various cDNA solutions were prepared as a dilution series (4 ng / µl; 2 ng / µl; 1 ng / µl; 0.5 ng / µl and 0.25 ng / µl). 15 µl of each of these cDNA solutions were mixed with 60 µl stock solution / primer mixture for the determination of GAPDH and distributed over 3 reaction vessels. A standard curve was also created for the 2-5 OAS PCR; the same dilutions that were used for the GAPDH standard curve were used. A PCR approach without cDNA served as a control. 15 µl of DEPC water were added to each 60 µl stock solution / primer mixture of 2-5 OAS and GAPDH, mixed and each divided into 3 reaction vessels. The amplification of the batches was carried out in the GeneAmp 5700 (2 min at 50 ° C; 10 min at 95 ° C, followed by 3 cycles at 15 s at 96 ° C and 2 min at 60 ° C; then 37 cycles at 15 s 95 ° C and 1 min at 60 ° C). The evaluation was carried out by determining the relative abundance of the 2-5 OAS gene in relation to the GAPDH reference. For this purpose, a standard curve was first drawn up by plotting the C _T values of the dilution series against the algorithm of the amount of cDNA in the PCR approach (RNA transcribed in ng) and determining the slope (s) of the straight line. The efficiency (E) of the PCR then results as follows: E = 10 ^{-1 / s} - 1. The relative abundance (X) of the 2-5 OAS cDNA species examined (Y) with respect to GAPDH is then: X = (1 + E _GAPDH ) ^C _T ^(GAPDH) / (1 + E _Y ) ^C _T ^(Y) . The numerical values were then standardized by setting the amount of intact skin cDNA from the 10-week-old BALB / c control animals or the C57BL / Ks control animals to 1.

The results of the experiment are shown in Table 2. For one thing, ve rified that the 2-5 OAS 1 expression in with the glucocorticoid Dexa wounds treated with methasone was significantly increased compared to normal healing Wounds from both 10 week old and young and old animals. IMP EXP however, a significant increase in expression was observed in all wounds observed against intact skin. This proves that wound healing with one type increased 2-5 OAS expression goes hand in hand. In contrast, in poorly healing Wounds in diabetic animals only showed a very slight increase in expression Wounds against intact skin are measured, while a clear in reduction in expression was observed in the control animals. It shows that the expression of 2-5 OAS in diabetic animals with poor wound healing is clearly misregulated and therefore the regulated expression of OAS and / or its binding partner RNAse L essential for the normal course the wound healing is.

Example 3 Analysis of the kinetics of expression of 2-5 OAS 1 during the Mouse wound healing using "TaqMan Analysis"

The kinetics of regulation of the expression of 2-5 OAS 1 during normal Wound healing of the mouse was determined using "TaqMan Analysis" in the GeneAmp5700 from Applied Biosystems examined. Normally healing day 1 wounds and intact skin was sheared from 6 untreated 10 week old BALB / c mice cut obtained as described in Example 1. The isolation of the RNA and the Subsequent TaqMan analysis was described as in the previous example ben performed. The numerical values were then standardized by the  Amount of intact skin cDNA from the 10 week old BALB / c control animals was set equal to 1. The relative changes in the murine 2-5 OAS 1 gene Expression during wound healing are summarized in Table 3. It It was found that within 24 hours of the wound, there was a Expression increases that lasts up to 5 days after wounding. to then there is a decrease in the 2-5 OAS 1 amount in the wound. This experiment shows that the differential expression over a long Period of wound healing is essential. Expression disorders and / or activity of the OAS and / or its RNAse L effector can therefore cause severe disorders in healing.

Example 4 Differential expression of the human 2-5 OAS-1 gene in human wounds

The normal healing wound should now be used to determine whether the wound in Examples 2 and 3 verified differential regulation of the expression of the as Wound-relevant identified 2-5 OAS genes can also be observed in humans leaves. For this purpose, 4 mm biopsies of intact skin as above were obtained from 6 patients described as well as 6 mm biopsies at times T = 1 h, 1d, 5d and 14d. The biopsies per time point were pooled and the mRNA as in described previous example isolated. Then the quantification was done using TaqMan analysis as described above, except that the frequency of human 2- 5 OAS 1 mRNA relative to cyclophilin (EMBL: Y00052) was determined. The Primers used for this experiment are cycliphilin primers 1: ATTGCTGACTGTGGACAACTCG (Seq ID No. 23), cyclophilin primer 2: AGAAGGAATGATCTGGTGGTTAAGA (Seq ID No. 24), hOAS primer 1: TCTCAGAAAT ACCCCAGCCA AA (SEQ ID No. 21) and hOAS Primer 2: (SEQ ID No. 22). The evaluation of the experiment is shown in Table 4. The Results prove that there is also a sharp increase in human wounds the hOAS1 expression comes, which lasts up to 14 days after wounding  

This experiment thus proves that the differential regulation of 2-5 OAS in Mammal wounds are essential for the course of wound healing, and that therefore 2-5 OAS and / or its effector RNAse L for diagnosis, prevention and / or treatment in wound healing or its disorders or in Er diseases of skin cells can be used.

Example 5 Differential expression of human 2-5 OAS1 in human ulcers

To show that the 2'-5'- identified as relevant for wound and skin disease Oligoadenylate synthetase in humans not only in normal wounds healing is regulated differentially but also in the case of a wound healing disorder Biopsies of patients with chronic venous ulcers were misregulated (Ulcera venosa) of both intact skin and the wound bed and Wound edge removed and examined for expression of 2-5 OAS 1. Of The biopsies of each group (intact skin, wound edge, wound bed) were taken from 6 subjects each pooled. The quantification of cDNAs relevant to wound healing was also carried out as described in Example 4. The results of the experiment are summarized in Table 5. In the wound margin of the ulcer, the Ä equivalent of the hyperproliferative epithelium of normally healing wounds that last 1 day after wounding, a slightly reduced expression compared to intact Skin can be measured while in normal healing wounds day-1 wounds a significant increase in 2-5 OAS-1 expression compared to intact skin was shown (Table 4). No increased 2-5 OAS mRNA amount observed. This proves that incorrect regulation of the 2-5 OAS Expression can lead to severe wound healing disorders. For prevention and / or treatment in wound healing or its disorders must therefore Expression of a 2-5 OAS and / or its effector RNAse L modulated be activated.  

Example 6 Differential regulation of the expression of human 2-5 OAS 1- Gens in lesional and non-lesional skin of psoriasis patients compared to intact skin of normal patients

It should now be verified on the basis of psoriasis patients that 2-5 OAS genes which can be used according to the invention play an important role not only in wound healing and wound healing disorders but also in other skin diseases. For this, psoriasis patients 4 mm punch biopsies were taken from both lesional and non-lesional skin as described in Example 5. As a control, biopsies of intact skin were taken from healthy volunteers. The mRNA was isolated from the individual biopsies as described below by embedding the biopsies in tissue freezing medium (Jung), crushing the biopsy using a microtome and then mRNA isolation using Dynabeads-Oligo dT (Dynal). The shredded biopsies are first suspended in lysis buffer and then homogenized with the Polytron. In order to crush the genomic DNA, the solution is centrifuged off via Qia-Shredder Columns (Qiagen) and sheared several times in a syringe with a cannula. The Dynabeads were pretreated according to the manufacturer's instructions and (250 µl of the stock suspension) mixed with the lysis homogenate, incubated and washed (final volume 250 µl). The suspension was then divided into a portion of 240 ul and 10 ul (as a control). The following components were mixed for the first strand synthesis: 20 ul 10 × TaqMan RT buffer, 44 ul 25 mM MgCl ₂ , 40 ul dNTP mix (2.5 mM / dNTP), 87 ul DEPC-H ₂ O, 4 ul RNase inhibitor (20 U / µl) and 5 µl MultiSc ribe Transcriptase (50 U / µl). 195 μl of the reaction mix were then added to the 240 μl batch and 20 μl to the control batch, mixed and incubated at 48 ° C. for 45 min. The Dynabeads were then pelleted in the magnetic stand and the supernatant removed. Then 20 μl of Tris-HCl buffer were added and the suspension was incubated at 95 ° C. for 1 min. The Dynabeads were immediately pelleted in the magnetic stand and the mRNA in the supernatant was removed. The cDNA / Dyanbeads were then washed 3 × with TE buffer. For the second strand synthesis, the cDNA / Dynabeads were washed 2 × in 1 × EcoPol buffer (NEB) and a solution of the following components was added: 23 μl 10 × EcoPol buffer; 4.6 µl dNTP mix (25 mM / dNTP); 11.5 µl of random hexamers; 118.7 µl DEPC-H ₂ O. The suspension was briefly mixed using the vortex mixer and then 9.2 µl Klenow fragment (5 U / µl) added. 200 μl of this solution were added to the batch, 20 μl to the control batch, and the suspensions were incubated at 37 ° C. for 1 h. The DNA was then melted at 94 ° C. for 1 min and the Dynabeads pelleted in the magnetic stand. The supernatant was transferred to a new reaction vessel and the enzyme was inactivated at 75 ° C. for 10 min. The sense DNA strands contained in the supernatant were then used for the Taq Man analysis.

The TaqMan analysis was carried out as described in Example 5, with the amount of GAPDH used to calculate the relative abundance of each mRNA species in the individual biopsies was used. Because from the skin biopsies of psoriasis patients, especially from lesional skin, are a lot greater total amount of mRNA can be isolated than healthy Pro from intact skin bound, normalization to equal amounts of mRNA is necessary, the Amount of GAPDH mRNA (primers used: hGAPDH primer 1: CCTCCCCTCTTCAAGGGTCTA (SEQ ID No. 19); hGAPDH primer 2: AGGAGTAAGACCCCTGGACCA (SEQ ID No. 20)) as housekeeping gene as Markers for the total amount of mRNA was adopted.

A total of 4 biopsies of intact skin from healthy subjects were analyzed, and 8 biopsies each of Psoria's lesional and non-lesional skin sispatienten. The abundances of the individual groups (intact skin, lesional Skin, non-lesional skin) were then added to the total of the abundances of the cDNAs measured on a microtiter plate. The averages of the Results are shown in Table 6. It becomes clear that a statistically sig significant (P <0.008, paired t-test) 3-fold increase in 2-5 OAS 1 expression  in lesional skin of 8 psoriasis patients compared to normal skin because the same patient can be observed. Furthermore, these results show a statistically significant (P <0.077, paired t-test), 4-fold increased amount of 2-5 OAS 1 mRNA in lesional skin of psoriasis patients compared to intact Skin of healthy subjects. This illustrates that this gene is misregulated or its direct interaction partner RNAse L lead to skin diseases can and that therefore the genes 2-5 OAS and RNAse L together or separately for prevention and / or treatment and / or Diagnosis of skin diseases are suitable. In the case of psoriasis patients to modulate the expression and / or activity of 2-5 OAS and / or RNAse L, the expression or activity of 2-5 OAS should preferably be inhibited. Be The modulation in the skin, in particular the lesional skin of the skin, is preferred Psoriasis patients.

The relationship between the incorrect regulation of the 2-5 OAS gene and a pathophysiological parameter of psoriasis disease become. The conductivity of the skin (a measure of the moisture of the skin) and with the 2-5 OAS 1 expression of the Gens compared in this area of skin. The skin's conductivity has been reduced Use of a corneometer according to the manufacturer (Courage and Khazaka Electronics). It was found that a statistically sig significant negative correlation (P = 0.0101, Pearson Product Moment Correlation) between conductivity of the biopsy examined and the 2-5 OAS 1- Expression can be observed: in very dry biopsies of lesional psoriasis Skin with low conductivity became a correspondingly high 2-5 OAS 1 mRNA Expression measured while in wetter skin, i.e. H. in the inconspicuous skin of the Psoriasis patients and healthy subjects in intact skin are significantly lower re 2-5 OAS expression is detectable. This verifies the relevance of the 2-5 OAS Expression for the pathogenesis of skin diseases.  

The results thus prove that 2-5 OAS and / or its effector RNAse L for Diagnosis, prevention and / or treatment in wound healing or its Disorders or diseases of skin cells can be used.  

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

SEQUENCE LISTING

Claims (20)

1. Use of at least one 2'-5'-oligoadenylate synthetase polypeptide according to SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 9 to SEQ ID No. 10 and / or RNAse L polypeptide according to SEQ ID No. 11 to SEQ ID No. 12 or functional variants thereof and / or nucleic acids encoding them or variants thereof, or a a 2'-5'-oligoadenylate synthetase poly peptide according to SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 9 to SEQ ID No. 10 and / or RNAse L polypeptide according to SEQ ID No. 11 to SEQ ID No. 12 or functional variants thereof and / or one encoding it Cell expressing nucleic acids or variants thereof for diagnosis, Prevention and / or treatment of diseases of skin cells in which Wound healing and / or their pathological disorders and their use for the identification of pharmacologically active substances. 2. Use according to claim 1, characterized in that the crane skin cell psoriasis. 3. Use according to claim 1, characterized in that the crane due to a deficiency in the 2'-5'-oligoadenylate synthetase mRNA is marked. 4. Use according to claim 1 or 3, characterized in that the pathological disorder of wound healing is a skin ulcer. 5. Use according to one of claims 1 to 4, characterized in that the polypeptide is used in the form of a fusion protein. 6. Use according to one of claims 1 to 4, characterized in that the nucleic acid in the form of an expression vector, a knock-out  Gene construct or a gene therapy vector used becomes. 7. Use according to one of claims 1 to 6, characterized in that that the cells mentioned are autologous or heterologous cells is. 8. Use according to claim 7, characterized in that it is in the Cells is a skin cell. 9. Use of an antibody or antibody fragment, preferably a polyclonal or monoclonal antibody or antibody question ment for the analysis, diagnosis, prevention and / or treatment of crane skin cells, wound healing and / or their pathological disorders tion and its use for the identification of pharmacological active substances, characterized in that an antibody produzie render organism with a polypeptide according to any one of claims 1 to 5 is immunized. 10. Use of at least one polypeptide according to claim 1 to 4 or ses encoding nucleic acid, or a a polypeptide according to claim 1 to 4 or a cell expressing this encoding nucleic acid, or ei nes directed against a polypeptide according to claim 1 to 5 o that of an antibody fragment, optionally combined or together with suitable additives and auxiliary substances, for the production of a diagnosis cumulatively for the diagnosis of diseases of skin cells and / or in the Wound healing and / or their pathological disorders. 11. Use of a diagnostic agent for the diagnosis of diseases of Skin cells and / or wound healing and / or their pathological disturbances  stanchions, characterized in that at least one polypeptide according to An say 1 to 4 or nucleic acid encoding it, or a polypep tid according to claim 1 to 4 or a nucleic acid encoding this expri cell or against a polypeptide according to claim 1 to 5 ge directed antibody or an antibody fragment, optionally combi niert or used together with suitable additives and auxiliaries becomes 12. Use of at least one polypeptide according to claim 1 to 4 or ses encoding nucleic acid, or a a polypeptide according to claim 1 to 4 or a cell expressing this encoding nucleic acid, or ei nes directed against a polypeptide according to claim 1 to 5 o that of an antibody fragment, optionally combined or together with suitable additives and auxiliaries, for the manufacture of a pharmaceutical for the prevention and / or treatment of diseases of skin cells, wound healing and / or pathological disorders. 13. Use of a medicament for the prevention and / or treatment of Diseases of skin cells, wound healing and / or their morbid ten disorders, characterized in that at least one polypeptide after Claims 1 to 4 or this nucleic acid coding, or a a poly A peptide according to claims 1 to 4 or a nucleic acid encoding it expressing cell, or an against a polypeptide according to claim 1 to 5 Directed antibody or an antibody fragment, optionally combi niert or used together with suitable additives and auxiliaries becomes. 14. Use of at least one polypeptide according to claim 1 to 4 or the ses encoding nucleic acid, or a a polypeptide according to claim 1 to 4 or a cell expressing this encoding nucleic acid, or ei  nes directed against a polypeptide according to claim 1 to 5 o that of an antibody fragment, optionally combined or together with suitable additives and auxiliary substances, for the identification of pharmaco logically active substances related to skin disorders cells and / or wound healing and / or their pathological disorders. 15. Use according to claim 14, characterized in that the pharma ecologically active substances the activity of at least one polypeptide influence according to one of claims 1 to 5. 16. Use according to claim 14, characterized in that the pharma ecologically active substances express at least one nucleic acid influence according to one of claims 1 to 4. 17. Use according to one of claims 14 to 16, characterized in that that at least one polypeptide according to claims 1 to 5 on a solid phase is bound. 18. Use according to any one of claims 14 to 16, characterized that at least one is a polypeptide according to claims 1 to 5 or one of these coding nucleic acid-expressing cell is used. 19. Use of at least one polypeptide according to claim 1 to 5 or ses coding nucleic acid, or at least one antibody according to An saying 9 for the production of an array fixed on a carrier material Analysis related to diseases of skin cells and / or of Wound healing and / or their pathological disorders.   20. Use of an array prepared according to claim 19 for analysis in Connection with diseases of skin cells, wound healing and / or their pathological disorders.