WO2010130073A1 - Itgb4bp and derivative thereof used in treatment and/or prevention of hyperplastic scar and fibrosis - Google Patents

Abstract

Provided are an integrin beta 4 binding protein, derivatives, and an expression vector thereof, as well as the use in preparation of the medicament or kit for prevention and/or treatment of hyperplastic scar or fibrosis, wherein the amino acid sequence of the integrin beta 4 binding protein is SEQ ID NO 1, and the derivatives thereof are protein derivatives which retain the binding part and functional part thereof.

Description

 ITGB4BP and its derivatives are used to prevent and/or treat hypertrophic scars and fibrotic lesions

 The present invention relates to novel pharmaceutical uses of known materials. Specifically, the present invention relates to a β4 integrin binding protein (Integrin beta 4 binding protein, ITGB4BP) and a derivative thereof, and a recombinant expression vector expressing ITGB4BP and a derivative thereof for preventing and/or treating hypertrophic scar and fibrotic lesions New application in . Background technique

 Fibrotic diseases are a type of lesion that causes tissue hyperplasia, hardening, and scar formation due to excessive deposition of extracellular matrix (ECM) such as collagen. Long-term stimulation of chronic inflammation, such as persistent infections, autoimmune diseases, allergic reactions, chemical damage, radiation and tissue damage, causes fibroblasts to differentiate into myofibroblasts, causing massive connective tissue deposition to form fibrosis, resulting in loss of organ function until Death (1-3). Hypertrophic scars are a type of fibrotic disease caused by fibrotic hyperplasia caused by heat or other forms of deep dermis, often causing serious damage to the patient's aesthetics and function (4). In the mechanism of hypertrophic scar-forming cell differentiation, we found that the P311 gene (GenBank ID: hsu36189) is closely related to it (5), but its specific mechanism is rarely reported in the literature.

The coding gene for P311 (also known as PTZ17, pentylenetetrazol 17) was first discovered by Matthieu in 1993 in brain tissue of embryonic rats (6). The P311 gene is located on chromosome 5 of ORF13, and its complete gene is about 2025 bp in length and contains three open reading frames, but only the first reading frame encodes a protein of P311 containing 68 amino acids with a molecular weight of 8KD. It is highly conserved between humans and mice. In addition, a highly conserved PEST domain (region rich in Pro, Glu, Ser and Thr) is present at the N-terminus of human, mouse and chicken P311 (7). P311 is widely expressed in a variety of tissues, including high expression in brain tissue, especially in the late embryonic development, and in adult cerebellum, hippocampus and olfactory bulb. It is also expressed in the liver, spleen, kidney, eye, heart and other organs and bone marrow. Tissue cells such as mesenchymal cells, fibroblasts, and myofibroblasts (6), and Pan and Fujitani, Taylor et al. also confirmed that the protein can be expressed in the cytoplasm and nucleus of myofibroblasts and nerve cells, respectively. P311 is a biological cytokine of the body that participates in cell differentiation, regulates other protein/gene transcription, wound repair, tumorigenesis, etc. Or abnormal biological activity (6-9).

 We used yeast two-hybrid technique to screen the coding sequence of a protein that interacts with P311 in an adult liver cDNA library (BD Clontech, USA). After bioinformatics analysis, resilience verification and laser confocal colocalization detection, we obtained a The gene of interest, which encodes a protein: Integrin beta 4 binding protein (ITGB4BP, SEQ ID NO 1 ) (10). Studies suggest that this protein plays an important role in cytoskeletal formation and apoptosis, and may play an important role in the differentiation of hypertrophic scar fibroblasts. The following review of the related functions of this protein at home and abroad is reviewed.

 1. ITGB4BP basic features

 1.1 The coding gene of ITGB4BP:

 Francesca Sanvito is equivalent to the discovery of the ITGB4BP gene (SEQ ID NO 2, NCBI Genebank accession number NM-002212) by the fluorescence in situ hybridization technique in 1998. The long arm 20ql l.2 region of chromosome 20 has a full length of 1108 bp. The open reading frame contains 735 nucleotides (11). The ITGB4BP gene has seven exons and six introns, and the 5' end of the gene lacks the TATA promoter binding region, CpG island (12). The ITGB4BP gene is sequence-conserved in eukaryotic cells, with 72% homology in mammals and yeast, with 85 percent sequence similar (13). The ITGB4BP gene is found in vertebrate animals such as human, mouse, rat, and Xenopus laevis. The presence of this gene is also found in invertebrates such as fruit flies, squid, and soft mites, and the domain of the gene is breast-feeding. Animals and invertebrates are highly conserved (14).

 1.2 ITGB4BP encoded protein:

 ITGB4BP protein (SEQ ID NO l, Swiss-Prot: P56537.1, [GI: 3122258]) Also known as EIF6, p27BBP, CAB, EIF3A, etc., was discovered by Biffo in 1997, Biffo et al. used yeast two-hybrid technique to study integrin When β4 is linked to hemidesmosome formation, it was found that a protein interacts with β4, hence the name of Integrin beta 4 binding protein (ITGB4BP). The ITGB4BP protein contains 245 amino acids with a molecular weight of 27 kd (15). This protein mediates signal transduction through phosphorylation/dephosphorylation under the action of protein kinase C, but its specific mechanism remains unclear (12, 16). The existing literature on ITGB4BP focuses on ITGB4BP and cytoskeletal formation, cancer cell differentiation, protein synthesis and migration.

1.3 ITGB4BP expression and distribution: The ITGB4BP protein is widely distributed and can be expressed in different tissues, or in different cells of the same tissue, or in different cycles of the same cell. Studies have confirmed that ITGB4BP protein is expressed in epithelial cells, fibroblasts, tumor cells, activated T cells, activated mast cells, and muscle tissue fibers, and studies have confirmed that as long as the expression of χ6β4 integrin cells express ITGB4BP ( 17) ₀ ITGB4BP protein is expressed in the cytoplasm (near the middle filament) and nucleus (mainly in the periphery of the nucleolus) (18). During the cell proliferation phase, the expression of ITGB4BP is up-regulated (19). Therefore, ITGB4BP protein and cytoskeleton can be suggested. Cell proliferation is related.

 2. The biological function of ITGB4BP

 2.1 Participation in the regulation of protein synthesis:

 During the growth and differentiation of eukaryotic cells, most of the genes in the genome are not transcribed, only a few genes can be expressed. These genes are processed by transcription and post-transcription to become mature mRNA and ribosomes in the cytoplasm. Translation into a polypeptide chain to form the protein required by the cell. There are many levels of regulation of mRNA translation, and the regulation of the initial stage of translation of eukaryotes is a very important step. At the initial stage of eukaryotic translation, ITGB4BP depolymerizes from the ribosomal 60s subunit, promoting the binding of the 40s subunit to the 60s subunit to form the 80s subunit, thereby initiating translation of the protein (20-23). A laboratory knockout of the ITGB4BP gene revealed a loss of the ribosomal 60s subunit, suggesting that it also plays an important role in the formation of ribosomal 60s (18). And in response to extracellular signals, ITGB4BP is involved in the translation of regulatory proteins as the earliest eukaryotic translation initiation factor (24). On the other hand, ITGB4BP regulates protein synthesis and is also involved in the regulation of microRNAs. ITGB4BP binds to RISC (RNA-induced silencing complex), specifically promotes the action of the corresponding microRNA, interferes with mRNA formation at the transcriptional level, inhibits the expression of its corresponding target protein, and vice versa when ITB4BP is knocked out. The microRNA inhibits the expression of the target protein and promotes the expression of the target protein (25 26).

 2.2 Participate in regulating cell adhesion and cytoskeletal formation:

The integrin family is an adhesion molecule receptor that is involved in mediating the interaction between cells and extracellular matrices, cells and cells. The adhesion between cells has a zygosity, a desmosome junction, a hemidesmosome junction, etc. The extracellular matrix of the hemidesmosome is mainly laminin, and the β4 integrin is a laminin receptor, so it is associated with desmosome and half. There is a close relationship between the formation of desmosomes. ITGB4BP is a β4 integrin-binding protein that binds to the functional domain of the intracellular domain of the intermediate filament and α6β4 integrin as a bridge between the two to mediate the formation of hemidesmosomes and mediate cell adhesion (15). In addition, ITGB4BP still exists In the nuclear matrix, the formation of the cytoskeleton is involved through the connection with the cytoskeletal structure of the nuclear matrix or intermediate filament. Experiments have shown that ITGB4BP is highly expressed on intermediate filaments and nuclear substrates (17). And ITGB4BP can also regulate the expression of β-catenin in the Wnt signaling pathway, thereby affecting the cytoskeleton (27).

 2.3 Relationship with tumorigenesis and metastasis:

 The most obvious features of tumors are abnormal cell proliferation, inhibition of apoptosis, and easy metastasis. Experiments have confirmed that ITGB4BP is involved in cell proliferation as a translational regulator. Subcellular proteomic analysis of the nuclear matrix components of liver cancer cells before and after differentiation induction revealed that ITGB4BP is highly expressed in differentiated cells, suggesting that it may be involved in cancer cell differentiation (28). ITGB4BP can be detected in normal mucosal cells, but overexpressed in cancer cells, such as upregulation of ITGB4BP in colorectal cancer (19), and is particularly evident in lymph node metastases (29), suggesting a high expression of ITGB4BP Participated in the proliferation and metastasis of tumors.

 It can be seen from the above that ITGB4BP plays an important role in ribosome synthesis, cytoskeleton and tumor proliferation, differentiation and metastasis, but there are few related literatures on ITGB4BP protein, and its role and function in fibrosis-related diseases. It has not been reported yet, so its function and role in fibrosis needs further study. Summary of the invention

 The inventors found that the P311 gene is closely related to the mechanism of differentiation of hypertrophic scar fibroblasts. At present, the research on the function and mechanism of P311 gene is still unclear. The inventors used the yeast two-hybrid technique to screen the coding sequence of a protein that interacts with P311 in an adult liver cDNA library (BD Clontech, USA), and further identified it. The protein encoded by the coding sequence was determined to be an integrin beta 4 binding protein (ITGB4BP, SEQ ID NO 1, Swiss-Prot: P56537.1, [GI: 3122258]). On the basis of this, the present inventors conducted a series of studies, demonstrating for the first time that ITGB4BP plays an important role in the differentiation process of hypertrophic scar fibroblasts, and completed the present invention.

In particular, the present invention relates to the use of a β4 integrin-binding protein (ITGB4BP) and a derivative thereof, or a recombinant expression vector expressing ITGB4BP and a derivative thereof for preventing and/or treating hypertrophic scar or fibrotic lesions. Β4 integrin binding protein (ITGB4BP) and its derivatives, or recombinant expression vectors expressing ITGB4BP and its derivatives, for the preparation of a drug or kit for the prevention and/or treatment of hypertrophic scar or fibrotic lesions, wherein the amino acid of ITGB4BP The sequence is SEQ ID NO 1, derived from human, and the ITGB4BP derivative is a protein derivative that retains the binding site and functional site of ITGB4BP (SEQ ID NO 1). Wherein the pharmaceutical composition or kit comprises a therapeutically effective amount of ITGB4BP or a derivative thereof, or a recombinant gene expression vector expressing ITGB4BP or a derivative thereof, and a pharmaceutically acceptable excipient or carrier. Preferably, the subject is a human or an animal, such as a mammal.

 Specifically, the present invention provides the following aspects:

 In the first aspect of the present invention, the present invention screens a recombinant vector pACT2-ITGB4BP carrying the ITGB4BP gene (SEQ ID NO 2) in an adult liver cDNA library, and based on this, clones the clone by conventional gene cloning technology. The recombinant shuttle plasmid pShutlle-CMV-ITGB4BP-EGFP was encoded by the ITGB4BP gene and the green fluorescent protein gene EGFP, and then the ITGB4BP-EGFP was recombined into the adenoviral vector pAdEasy-1 by gene homologous recombination to obtain the recombinant adenovirus expression vector pAdEasy- ITGB4BP-EGFP, and packaged into adenoviral particles, used for the identification of ITGB4BP protein function and ITGB4BP gene function. Furthermore, the lentiviral vector pFIV-H 1 /U6-copGFP-ITGB4BP NAi related to the ITGB4BP gene was further cloned for RNA interference experiments for the function of ITGB4BP protein and the identification of ITGB4BP gene function.

 In the second aspect of the invention, the present invention separates normal skin fibroblasts and hypertrophic scar fibroblasts, and establishes a scar cell model. Furthermore, the expression level of ITGB4BP in the above two cells was identified, and ITGB4BP was found to be lowly expressed in hypertrophic scar fibroblasts (see Fig. 1), indicating that low expression of ITGB4BP aggravated fibrosis of hypertrophic scar.

 In the third aspect of the present invention, the present invention detects the expression of fibrosis-related markers TGF-βΙ (transforming growth factor βΐ) and ΜΜΡ9 (matrix metalloproteinase 9) by inhibiting the expression of ITGB4BP in hypertrophic scar fibroblasts by RNAi technology. And the expression level of type I collagen. The results showed that the expression of TGF-βΙ, MMP9 and type I collagen was increased in the expression of fibrosis-related indicators after suppression of ITGB4BP expression at the mRNA level (see Figure 2), which significantly aggravated fibrosis.

In the fourth aspect of the invention, the present invention detects the expression levels of fibrosis-related indicators TGF-pi, MMP9 and type I collagen by increasing the expression of ITGB4BP in normal skin fibroblasts. The results indicate that fibrosis is associated with increased expression of ITGB4BP at the mRNA level. The expression levels of TGF-pi, MMP9 and type I collagen were significantly reduced (see Figure 3), inhibiting fibrosis.

 In a fifth aspect of the invention, the present invention detects the expression levels of myofibrillar surface markers α-SMA and fibrosis-related indicators TGF-βΙ and type I collagen by highly expressing ITGB4BP in hypertrophic scar fibroblasts. And inhibit the contraction of myofibroblasts using a three-dimensional culture system (FPCL) of fibroblasts. The results showed that the expression of α-SMA was decreased after high expression of ITGB4BP in hypertrophic scar fibroblasts compared with the negative control (see Figure 4), inhibiting the transformation of hypertrophic scar fibroblasts into myofibroblasts; The expression of TGF-pl, MMP9 and type I collagen was significantly inhibited (see Figures 5 and 6), and the contractility of myofibroblasts was decreased (see Figure 7).

 Therefore, the present invention demonstrates for the first time that ITGB4BP can inhibit the production of collagen by fibroblasts, fibroblasts, and myofibroblasts in an in vitro cell culture system (including type I and type III, etc., herein shows data on type I collagen, Data on type III collagen were shown); ITGB4BP was first demonstrated to inhibit contraction of myofibroblasts; ITGB4BP was found to down-regulate TGF-βΚ MMP9 expression. Therefore, the present invention concludes that in fibrotic diseases, low expression of ITGB4BP may be an important initiating factor regulating phenotypic transformation and functional changes of fibroblasts, and ITGB4BP may be a potent fibrosis. Related negative signal. Therefore, the high expression of ITGB4BP has obvious anti-fibrotic effect. Therefore, ITGB4BP and its gene expression vector can be used for the prevention and/or treatment of hypertrophic scar disease or fibrotic lesions.

 On the other hand, due to the highly conserved sequence of the ITGB4BP gene in eukaryotic cells, natural or genetically engineered protein derivatives that play a biological role in the binding site and functional site of ITGB4BP retain a conserved functional domain with similar functions. It can also be used to prevent and/or treat hypertrophic scar disease or fibrotic lesions. In particular, it has a binding site and a functional part of ITGB4BP (SEQ ID NO 1 ), and can inhibit the expression of fibrosis-related indicators such as TGF 1, MMP9 or type I collagen, or inhibit fibroblast surface markers after high expression. The expression of the ITGB4BP derivative of a-SMA is included in the scope of the present invention.

Accordingly, in a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition or kit for preventing and/or treating a hypertrophic scar disease or a fibrotic lesion, the pharmaceutical composition or kit comprising the treatment An effective amount of ITGB4BP or its derivative, or expression of ITGB4BP A recombinant gene expression vector of the same or a derivative thereof, and a pharmaceutically acceptable excipient or carrier.

 In another preferred embodiment of the invention, the pharmaceutical composition or kit further comprises an inhibitor of scar or fibrotic lesions currently known for use.

 Accordingly, the present invention also provides a method of preventing and/or treating hypertrophic scar and fibrotic lesions, the method comprising administering to a subject a therapeutically effective amount of ITGB4BP or a derivative thereof, or expressing ITGB4BP or a derivative thereof A recombinant gene expression vector, or a pharmaceutical composition or kit as described above, preferably, the subject is a human or an animal, such as a mammal.

 It will be understood by those skilled in the art that the pharmaceutical composition or kit may be administered topically to the scar or fibrosis, or may be administered by conventional methods such as oral administration, intravenous injection, intramuscular injection or the like. A person skilled in the art can determine the dose level of ITGB4BP or a derivative thereof, or a recombinant gene expression vector expressing ITGB4BP or a derivative thereof, and a pharmaceutical composition, respectively, by routine experimentation. It will be appreciated that the particular dosage level for any particular subject will depend on a variety of factors, including the subject's age, weight, general state of health, sex, diet, time of administration, route of administration and excretion rate, drug combination, and experience of treatment. The severity of the specific disease, etc. In the case of administering the pharmaceutical composition, ITGB4BP or a derivative thereof, or a recombinant gene expression vector expressing ITGB4BP or a derivative thereof may be administered at the same time or at intervals as other scar or fibrotic lesion inhibitors.

 The present invention also provides the use of an ITGB4BP protein or a derivative thereof, a recombinant gene expression vector expressing ITGB4BP or a derivative thereof, for the preparation of a medicament or kit for preventing and/or treating a hypertrophic scar disease or a fibrotic lesion.

 In the present invention, the hypertrophic scar includes, but is not limited to, skin burns such as burns, burns, cuts, chemical damage, and freezing injuries. The fibrotic lesion includes pathological fibrosis of various tissues and organs, such as, but not limited to, skin tissue fibrosis, pulmonary fibrosis, liver fibrosis, or renal fibrosis; and includes various types of fibrosis. Pathological contractions, such as, but not limited to, body surface and internal organs, pathological contractions of tissues, and the like.

Finally, the present invention also provides a method for obtaining ITGB4BP or a derivative thereof, which can be cloned into a suitable prokaryotic expression vector, eukaryotic expression vector such as yeast expression vector by genetic recombination technology by encoding a nucleotide encoding ITGB4BP or a derivative thereof. In the viral expression vector, a suitable recombinant expression vector is constructed, transformed or transfected into the corresponding host for recombinant expression, and finally subjected to a protein purification step to obtain ITGB4BP or a derivative thereof. In a preferred embodiment of the invention In the protocol, the method encodes a nucleotide encoding ITGB4BP into a viral expression vector, such as an adenovirus pAdEasy-1 vector, for recombinant expression.

 It should be noted by those skilled in the art that the ITGB4BP or the gene encoding the same according to the present invention is mainly derived from humans. DRAWINGS

 The above features and advantages of the present invention will become more apparent from the following detailed description of the appended claims.

 Figure ITGB4BP expression in normal skin and hypertrophic scar fibroblasts.

 Figure 2: Increased expression of TGF-βΚ MMP9 and type I collagen in culture supernatants after inhibition of ITGB4BP expression in hypertrophic scar fibroblasts. 2A: Screening for effective lentiviruses for interference with the ITGB4BP gene, 1#, 2# and 3# respectively represent lentivirus-FIV-Hl U6-copGFP-ITGB4BPR Ail#/2#/3#; 2B: in hypertrophic scar fibrils Detection of expression of TGF-pl, MMP9 and type I collagen in culture supernatant after inhibition of ITGB4BP expression in cells, * : Ρ = 0.0001, * * : = 0.005, cadaver = 0.03.

 Figure 3: Expression of TGF-pi, ΜΜΡ9 and type I collagen in cell culture supernatants after high expression of ITGB4BP in normal skin fibroblasts, *: household = 0.038, * *: corpse = 0.0005, female: Ρ = 0.007.

 Figure 4: Expression of a-SMA in cells after elevated expression of ITGB4BP in hypertrophic scar fibroblasts. 4A: mRNA level detection; 4B: protein level detection; 4C: protein level analysis, 1 is EGFP, 2 is ITGB4BP-EGFP.

 Figure 5: Expression of TGF-βΙ in cells after elevated expression of ITGB4BP in hypertrophic scar fibroblasts. 5A: mRNA level detection; 5B: protein level detection; 5C: protein level analysis, 1 is EGFP, 2 is ITGB4BP-EGFP.

 Figure 6: Expression of type I collagen in cells after elevated expression of ITGB4BP in hypertrophic scar fibroblasts at the mRNA level.

 Figure 7: Detection of contractile function of myofibroblasts after high expression of ITGB4BP in hypertrophic scar fibroblasts. 7A, 3D gel model, left image is ITGB4BP-EGFP, right image is EGFP; 7B, contraction index analysis, P=0.008, 1 is ITGB4BP-EGFP, 2 is EGFP.

Figure 8: Plasmid map. 8A, pEGFP-N2 _; 8B, pl8MD-18; 8C, pShuttle-CMV; 8D, pAdEasy-1; 8E, pFIV-Hl U6- copGFP. detailed description

 The invention is further illustrated by the following examples. However, it should be understood that the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the invention.

 The experimental apparatus and materials used in the present invention and their sources are shown in Table 1 below.

 Table 1. Experimental instruments and materials used in the present invention and their sources

 Name Source

 MyCyclear TY5533 type PCR instrument USA Bio-Rad company

 Mini Protein 3 cell Small Vertical Electrophoresis System USA Bio-Rad Company

 Portable UV Lamp USA Upland Corporation

 Gel Imaging Analysis System Gel Doc2000 USA Bio-Rad

 SDS-PAGE electrophoresis system USA Bio-Rad company

 Mini Trans-Blot Electric System US Bio-Rad

 CP225D electronic balance Germany Sartorius

 HH · W 21 · Cu600 electric heating constant temperature water tank Shanghai Medical Equipment No. 7 Factory

 LS-B50L vertical pressure steam sterilizer Shanghai Huaxian Medical Instrument Company

 LDR0.08-0.7G electric vacuum sterilizer Shandong Xinhua Medical Equipment Co., Ltd.

 DGF20003 electric blast drying oven Chongqing Test Equipment Factory

 HQ45 type constant temperature shaker

 High-speed desktop centrifuge -TGL-18C-C Shanghai Anting Instrument Factory

 SZK-202 Purification Workbench 蚌埠Insulation Material Factory

 HHB11.420-S type constant temperature incubator Shanghai Yuejin Medical Instrument Factory

 DU-640 UV Spectrophotometer United States Beckman

 CR21F low temperature high speed centrifuge Japan HITACHI company

 -80°C Low Temperature Refrigerator (HARRIS) USA HARRIS Company

 Milli-Q Biocel Water Meter USA Millipore Company

 Various pipettes USA Eppendorf

 DH-IIDNA Rotary Mixer Ningbo Xinzhi Biotechnology Co., Ltd.

F-3010 Fluorescence Spectrophotometer Japan Hitachi Company

 7500 real-time PCR instrument USA ABI company

680 type microplate reader US Bio-Rad pEGFP-N2 BD clontech, Inc. (6081-1) pITGB4BP-EGFP Plasmid constructed in our laboratory, see Example 1 for the construction method.

pAdEasy-1 plasmid, pShuttle-CMV plasmid United States Qbiogene

Escherichia coli DH5a, BJ5183 United States Qbiogene

Tryptone Sigma USA

Agarose Swiss Roche Company Bromophenol Blue Shanghai Sangon Company

 Goldview Nucleic Acid Dyes Beijing Saibai Company

Endonuclease required by the present invention, yTaq and other tool enzymes Dalian TaKaRa Company

Lipofectamine ²⁰⁰⁰ Dalian TaKaRa

Ampicillin, kanamycin Shanghai Huamei Company

Sequencing and Primer Synthesis Shanghai Boya Company

Mouse anti-human ITGB4BP polyclonal antibody Taiwan Abnova

Mouse anti-human α-SMA monoclonal antibody Wuhan Dr.

Mouse anti-human β-actin monoclonal antibody Wuhan Dr.

Mouse anti-human TGF-βΙ monoclonal antibody United States Peprotech

Peroxidase-labeled goat anti-mouse IgG Wuhan Dr.

 RIPA Lysis USA Santa Cruz

Chemiluminescent liquid United States Santa Cruz

 RIPA Shanghai Shenneng Company

 SDS Shanghai Xibao Company

 Tris HCl Shanghai Xibao Company

Acrylamide Shanghai Xibao Company

 Ν'Ν'-甲叉双acrylamide Shanghai Xibao Company

 PVDF membrane USA Millipore Corporation

Skim milk powder Inner Mongolia Yili Company

Six-well and 24-well plates Corning, USA

Trypsin USA Hyclone

Calf serum Chengdu Harry company

 DMEM USA Hyclone Corporation

75cm ² culture bottle Shanghai Wanhong Glass Instrument Factory

EP tube American Axygen company Primer synthesis Shanghai Invitrogen

 Real-time PCR Master Mix (SYBR Green) Japan Toyobo Corporation

 Reverse Transcription Kit AMV3.0 Dalian TaKaRa

 Tripure Swiss Roche

 DNA Ligation Kit USA MBI

 Plasmid extraction, DNA purification and recovery kit United States Omega

 Human MMP-9 ELISA Test Kit United States RB

 Human Type I Collagen ELISA Test Kit United States RB

 Human TGF-βΙ ELISA Test Kit United States RB

Example 1. Construction of shuttle vector

 1.1 Acquisition of target gene sequences

 This experiment was carried out using PEGFP-N2 and pITGB4BP-EGFP plasmid, pEGFP-N2 was purchased from clontech (Cat. No. 6081-1), and pITGB4BP-EGFP was a plasmid constructed in this laboratory. The pITGB4BP-EGFP was constructed by designing primers according to the sequence of the recombinant vector pACT2-ITGB4BP (liver cDNA library, BD Clontech, USA):

Upstream primer pITGB4BP-EGFP- oRI (insertion of coRI restriction site):

 5 '-CAGAATTCATGGCGGTCCGAGCTTCGTT-3 ';

Downstream primer pITGB4BP-EGFP- amffl (insert SamHI restriction site):

 5 '-CAGGATCCCGGTGAGGCTGTCAATGAGGGAAT-3 '. The PCR reaction was carried out using pACT2-ITGB4BP as a template, and the reagent was a product of TaKaRa. The reaction system is as follows:

 PACT2-ITGB4BP Ι μΐ (approximately ^g )

 l OxPCR buffer 5μ1

 dNTP (2.5 mmol/L) 4μ1

MgCl ₂ (25 mmol/L) 3μ1

 Upstream primer 0.5μ1

 Downstream primer 0.5μ1

 rTaq enzyme (5υ/μ1) 0.5μ1

ddH ₂ 0 35.5μ1

 Final volume 50μ1

Reaction conditions: Denaturation at 94 ° C for 4 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds for 30 cycles, and then 72 ° C for 10 minutes, to obtain a target fragment of about 750 bp ITGB4BP. First, according to the PMD18-T vector ligation kit (TaKaRa), the PCR product is connected to In the pMD 18-T vector, a recombinant pMD18-T-ITGB4BP was obtained. Then, the PCR product of ITGB4BP and the green fluorescent protein expression vector pEGFP_N2 were digested with EcoRI and ^mffl, and the reaction conditions were as follows:

 pEGFP-N2 (or pMD 18-T-ITGB4BP ) 40μ1 (approx. 2 g ) ΙΟχΗ buffer 5μ1

 EcoRI 2.5μ1

 B mHl 2.5μ1

 Final volume 50μ1

Reaction conditions: 37 ° C reaction for 2 h. Then, electrophoresis was carried out using a 0.6% agarose gel, and the target strip was subjected to gelatinization and gel recovery. Glue recovery using DNA purification and recovery kit (purchased from the United States)

Omega), purify and recycle according to the instructions provided by the supplier. Finally get coRI and

BamHl double-cut the ITGB4BP fragment and the pEGFP-N2 linear plasmid fragment. Then use T4

DNA ligase (Dalian Takara Co., Ltd.) was connected to the gel recovery product, and the reaction conditions were as follows: pEGFP-N2 after recovery 3.5 μl (about 0.5 μm _β ) recovered ITGB4BP 12 μl (about 1)

 10xT4 DNA ligation buffer 2μ1

 Τ4 DNA ligase 2.5μ1

 Final volume 20μ1

Reaction conditions: 16 ° C overnight. The constructed recombinant vector was named pITGB4BP-EGFP. After transforming the ligated product into competent E. coli DH5 (x, the positive clone was screened by kanamycin resistance, single colony was picked for amplification, the plasmid was extracted and the product was double-digested with coRI+^zmffl, and used.

The results of digestion were identified by 0.8% agarose gel electrophoresis. So far, the pITGB4BP-EGFP vector was constructed.

 Then, Bglll and Notl (both purchased from Dalian Takara Co., Ltd.) were used to digest pEGFP-N2 and pITGB4BP-EGFP to obtain the target fragment EGFP (about 796 bp in length) and

ITGB4BP-EGFP (about 1512 bp in length) was constructed into pShuttle-CMV (Abiogene, USA, about 7.5 kb in length) treated with the same double digestion. The respective digestion systems of pEGFP-N2, pITGB4BP-EGFP and pShuttle-CMV are as follows:

_PEGFP -N2 (or _PITGB4BP - _EGF P or _{28 1} (approximately ₂ ) pShuttle-CMV ) ^{μ J μ}

 ΙΟχΗ buffer 4μ1

 0.1% BS Α 4μ1

 NotI, Bgl ll each 2μ1

 Final volume 40μ1

Reaction conditions: The reaction was carried out at 37 ° C for 2 h, and electrophoresis was carried out on a 0.6% agarose gel, and the target strip was subjected to gelatinization and gel recovery. Glue recovery using DNA purification and recovery kit (purchased from Omega, USA) Division), the method is carried out according to the instructions provided by the supplier. Finally, EGFP, ITGB4BP-EGFP fragment and pShuttle-CMV linear plasmid fragment double-digested with Bgl ll and Notl were obtained.

1.2 Shuttle carrier connection

 The target fragment EGFP and ITGB4BP-EGFP obtained in the previous step were ligated to the same digested and recovered plasmid pShuttle-CMV by T4 DNA ligase, respectively, thereby obtaining a shuttle plasmid vector containing the desired fragment. The respective connection reaction systems of the two are as follows:

 Recovered pShuttle-CMV 3.5μ1 (approx. 0.5 g) EGFP or ITGB4BP-EGFP 12μ1 (about l g) after recovery

 10xT4 DNA ligation buffer 2μ1

 Τ4 DNA ligase 2.5μ1

 Final volume 20μ1

 Reaction conditions: 16 ° C overnight.

 Transformation and amplification of ligation products: The ligation product was transformed into competent E. coli DH5a cells prepared by the calcium chloride method according to a conventional E. coli transformation method well known in the art, and positive clones were screened using kanamycin resistance, and Positive clones were subjected to plasmid amplification, identification and extraction of recombinant plasmids.

 1.3 Identification of the shuttle vector after construction

 The ligation product obtained in the previous step was identified by Xho I (purchased from Dalian Takara Co., Ltd.), and the recombinant shuttle plasmid vectors were successfully designated as pShuttle-CMV-ITGB4BP-EGFP and pShuttle-CMV-EGFP, respectively. The identified enzyme digestion system is as follows:

pShuttle-CMV-ITGB4BP-EGFP or pShuttle-CMV-EGFP 5μ1 (approximately 0.2μ _β )

ΙΟχΗ buffer Ιμΐ

ddH ₂ 0 3.5μ1

 Xho I 0.5μ1

 Final volume ΙΟμΙ

 Reaction conditions: 37 ° C reaction for 2 h, 0.6% agarose gel electrophoresis to identify whether the construction was successful. Example 2. Homologous recombination of adenovirus

The shuttle vector successfully constructed in Example 1 was digested with Pme I, and then transformed into the competent Escherichia coli BJ5183 containing the pAdEasy-Ι plasmid (purchased from Qbiogene, USA) prepared by the calcium chloride method. , complete homologous recombination of adenovirus. Specific operation The content is as follows:

 2.1 Linearization of the shuttle plasmid:

 The successfully constructed shuttle plasmids pShuttle-CMV-ITGB4BP-EGFP and pShuttle-CMV-EGFP were digested and recovered with Pme I (purchased from NEB, UK). The respective enzyme digestion systems are as follows - pShuttle-CMV-ITGB4BP-EGFP or pShuttle-CMV-EGFP 18 μΐ (approximately 0.5)

NEB buffer 4 5 μ1

 lOOxBSA 0.5 μΐ

ddH ₂ 0 25.5 μΐ

 Pme I 1 μΐ

 Final volume 50 μΐ

 Reaction conditions: After reacting at 37 ° C for 2 h, 0.6% agarose gel electrophoresis, gelation and gel recovery (method same as 1.1 in Example 1).

2.2 homologous recombination of adenovirus:

 The linearized shuttle plasmids were separately transformed into Escherichia coli BJ5183 prepared from calcium chloride containing the adenovirus pAdEasy-Ι vector by a conventional E. coli transformation method well known in the art. Positive colonies were screened in kanamycin resistant solid medium. Small and medium-sized colonies were picked from the grown large, medium and small colonies. After amplification, the plasmid was extracted and identified by restriction enzyme digestion with Pac l (purchased from NEB, UK). The recombinant adenoviral vectors were named separately. For pAdEasy-ITGB4BP-EGFP and pAdEasy-EGFP. The identified enzyme digestion systems are as follows: pAdEasy-ITGB4BP-EGFP or pAdEasy-EGFP 4μ1 (approximately 0.1)

 NEB buffer 1 2μ1

 lOOxBSA 0.2μ1

ddH ₂ 0 13.5μ1

 Pad 0.3μ1

 Final volume 20μ1

 Reaction conditions: After reaction at 37 ° C for 1 h, 0.6% agarose gel electrophoresis, Goldview nucleic acid staining showed bands of 23 kb and 3.0 kb or 23 kb and 4.5 kb, respectively, indicating that the adenoviral vector was successfully recombined. Example 3. Packaging and amplification of adenovirus,

 3.1 Adenovirus vector transfection HEK293 cell packaging adenovirus

 3.1.1 Linearization of adenoviral vectors:

Recombinant adenoviral vector pAdEasy-ITGB4BP-EGFP was digested with Pac I pAdEasy-EGFP was recovered. The enzyme digestion system is as follows:

 pAdEasy-ITGB4BP-EGFP or pAdEasy-EGFP 48μ1 (approx. 1.(^g)

 NEB buffer 1 8μ1

 lOOxBSA 0.8μ1

ddH ₂ 0 21.7μ1

 Pad 1.5μ1

 Final volume 80μ1

 Reaction conditions: After reacting at 37 ° C for 1 h, 0.6% agarose gel electrophoresis, Goldview nucleic acid dye staining showed 23 kb and 3.0 kb or 23 kb and 4.5 kb bands, respectively, for the above 23 kb bands (respectively including recombination The target genes pAdEasy-ITGB4BP-EGFP and pAdEasy-EGFP) were subjected to gel recovery (the same method as in Example 1 1.1).

3.1.2 Packaging of adenovirus:

3× ^{10 5} cell HEK293 cells (purchased from the Chinese Academy of Sciences cell bank) were inoculated into a six-well plate (Coming, USA), and transfection was started when the cells reached 80% confluence. The transfection method was as follows: 8 g of adenoviral plasmid linearized in 3.1.1 was mixed with 250 μl of anti-DMEM (ie, antibiotic-free DMEM) (purchased from Hyclone, USA), room temperature, 5 min; 20 μl lipid The plastid was gently mixed with 480μ1 anti-DMEM, and divided into 2 equal portions at room temperature for 5 minutes. The mixture of the previous two steps was mixed and allowed to stand at room temperature for 20 min. After washing 293 cells in the six-well plate twice with PBS, the plasmid lipid was added. The mixture of plastids; freshly anti-DMEM (containing 10% calf serum), 2 ml per well, after 37 hrs in C0 ₂ incubator for 4 hr. After 24 hours of culture, the intracellular fluorescence expression was observed.

 3.2 Collection and amplification of adenovirus:

 On the sixth day after transfection of HEK293 cells, the fluorescence was strong and the cells were completely suspended. The cells were blown down with sterile tips and collected in EP tubes (purchased from Axygen, USA) at -80 °C. The supernatant obtained by repeated freezing and thawing for 3 times at 37 ° C, 12,000 rpm, and centrifugation at 4 ° C for 15 min was the primary virus solution, and named Ad-ITGB4BP-EGFP and Ad-EGFP, respectively. The virus solution was then continued to infect 293 cells, and the 3-4rd generation virus solution was collected by the same method. Example 4. Construction and packaging of lentiviral interference vector

The lentiviral interference vector pFIV-Hl U6-copGFP-ITGB4BPRNAi of the ITGB4BP gene required in this example was purchased from Chongqing Jinmai Biotechnology Co., Ltd. (Address: 3-11, Jindihui, Tianxingqiao, Shapingba District, Chongqing) as a negative control. Interference sequence Also purchased from the company. The lentiviruses obtained by packaging the above two transfected 293FT cells were named as: lentivirus-FIV-Hl/U6-copGFP-ITGB4BPRNAi 1#/2#/3# and a negative control.

 The candidate fragments selected for RNA interference screening are:

ITGB4BP R Ai 1#-1: AAAG GGGCTGGTGC ATCCC AAGA

ITGB4BP RNAi 1 #-2: AAAA TCTTGGGATGC ACCAGCCC

ITGB4BP R Ai2#- 1: AAAG GGCTC AGAGAACTTCTAC A

ITGB4BP RNAi2#-2: AAAA TGTAGAAGTTCTCTGAGCC

ITGB4BP RNAi3#-l: AAAG CAGCCTCCCAGACACAGTG

ITGB4BP RNAi3#-2: AAAA CACTGTGTCTGGGAGGCTG

Negative control RNAi- 1: AAAG TCGACTCAGTCGGGTGGCA

Negative control R Ai-2: AAAA CTGCTATCGAGCCTGGCGA

 Construction of lentiviral vector according to SBI company pFIV--Hl/U6-copGFP siRNA kit

(Cat: #SI111A-1) Operation manual.

 4.1 Construction of lentiviral vector pFIV-Hl/U6-copGFP-ITGB4BPRNAi

 The above candidate fragments were annealed in the following ratios. The reaction system is as follows:

Upstream segment 2.5μ1 (approximately ?.5μ§) downstream segment 2·5μ1 (approximately 2,5μ _§ )

2 X annealing buffer 25.0μ1

ddH ₂ 0 20.0μ1

 Final volume 50.0μ1

 Reaction conditions: The reaction was carried out at 95 ° C for 5 minutes and then cooled to room temperature.

The successfully ligated fragment was then ligated to pFIV-Hl/U6-copGFP. The reaction system is as follows: Candidate fragment Ιμΐ (about 0.01μ _§ ) pFIV-Hl/U6-copGFP 2,5μ1 (about 0.5μ _§ )

10xT4 DNA Ligation Buffer Ιμΐ

 T4 DNA ligase Ιμΐ

ddH ₂ 0 4.5 μ 1

 Final volume 10 μ ΐ

 The reaction conditions were as follows: 16 ° C overnight.

 Transformation and amplification of ligation products: The ligation product was transformed into competent E. coli DH5 prepared by the calcium chloride method according to a conventional E. coli transformation method well known in the art (cells, positive clones were screened using ampicillin resistance, and positive Cloning is performed for plasmid amplification, identification and extraction of recombinant plasmids.

 4.2 Lentiviral interference vector pFIV-Hl U6-copGFP-ITGB4BPR Ai packaging

4×10 ⁵ cell 293FT cells (purchased from the Chinese Academy of Sciences cell bank) were inoculated into a 75 cm ² culture flask (Shanghai Wanhong Glass Instrument Factory), and transfection was started when the cells reached 70% confluence. Transfected party The method was as follows: 2.5 μg of envelope protein pVSV-G, 7.5 μg of packaging plasmid pFIV34N and 2 μg of the above successfully constructed expression plasmid were mixed in ddH20 (total volume 1095 μΐ), and then 155 μΐ 2Μ CaCl ₂ was added. Then slowly add 1250 μΐ 2 X PBS and mix. After washing the 293FT cells twice with PBS, the above mixture was added; after incubating at 37 ° C for 7 hr in a C0 ₂ incubator, fresh anti-DMEM (10% calf serum) was replaced, 10 ml per bottle. After 48 hours of culture, the cells showed green fluorescence. The cell supernatant was collected, centrifuged at 3000 rpm for 5 min at 4 ° C, and the supernatant was filtered with a 0.45 μm PVDF membrane to provide the required lentiviral solution. Example 5. Isolation and culture of primary dermal fibroblasts

 Normal foreskin fibroblasts were obtained from the Department of Urology, Chongqing Southwest Hospital (with written consent from the patient). The hypertrophic scar fibroblasts were obtained from the burn department of Chongqing Southwest Hospital, and were collected from patients within one year after burn (with written consent of the patient).

The methods for isolation and culture of primary skin and hypertrophic scar fibroblasts are as follows: Place the tissue block in a sterile Petri dish, rinse it three times with PBS, remove the epidermis and subcutaneous tissue with sterile scissors, and cut the dermal tissue; The crushed tissue was placed in a 25 ml conical flask, and added with 0.5% trypsin (purchased from Hyclone, USA) 10 ml, shaken at room temperature for 2 h; 10 ml of DMEM containing 10% calf serum (purchased from Chengdu Hari Company) was added. (purchased from Hyclone, USA) to terminate the digestion, filter the suspension through a sterile filter and discard the tissue fragments; centrifuge, 800 rpm l0min, discard the supernatant, add 10ml DMEM containing 10% calf serum, wash twice, centrifuge again After discarding the supernatant, resuspend the cells in 10 ml DMEM containing 10% calf serum and transfer to a 75 cm ² culture flask (purchased from Shanghai Wanhong Glass Instrument Factory), 37 ° C, 5% C0 ₂ incubator The culture was carried out, and the medium was changed after 24 hours while removing the non-adherent cells. After the cells are completely over, they can be subcultured. The 6th to 10th generation fibroblasts were selected for the experiment.

5.1 Normal skin and hypertrophic scar fibroblast RNA extraction

The specific steps are as follows: Take the 8th generation fibroblasts (six-plate culture), discard the medium; add 200μ1 Tripure (purchased from Roche, Switzerland, catalog number: 11667157001) per well, and place on the ice box for 5min. After scraping all the cells and collecting them; add 40 μl of trichloromethane in a volume ratio of 1:5 (trichloromethane: Tripure, v/v), shake vigorously for 15 sec, place lOmin on the ice box; then add 200 μl Tripure per well. Place on the ice box for 5 min; 4 ° C, 12000 g, centrifuge for 15 min; transfer the upper aqueous phase, add an equal volume of isopropanol, mix, place on the ice box lOmin; 4 °C, 12000g, centrifuge lOmin; discard the supernatant, add 75% ethanol (1% diluted with DEPC water) 500μ1 wash precipitate; 4 ° C, 7500g, centrifuge for 5min _; 1%. The DEPC water-dissolved precipitate was RNA; the concentration of RNA was determined and frozen at -80 °C.

 5.2 Reverse transcription reaction after RNA extraction

 The reverse transcription reaction was carried out by using the NA extracted in the previous step as a template. The reverse transcription reaction system is as follows:

 RNA extracted in 5.1 about l g

 AMV reverse transcriptase ΙμΙ

 RNase inhibitor 0.5μ1

 dNTP mixture 2μ1

 lOxRT buffer 2μ1

 Oligo dT-linker primer Ιμΐ

MgCl ₂ 4μ1

 RNA

ddH ₂ 0 completion to 20μ1 final volume 20μ1

 Reaction conditions: 50 ° C for 30 min, 99 ° C for 5 min, 5 ° C for 5 min, 4 ° C;

 5.3 Real-time PCR detection (Real-time PCR)

 The real-time PCR reaction system is as follows:

 RT reactant 2μ1

 SYBR Green Real Time PCR Master Mix ΙΟμΙ

 Forward primer (ΙΟηΜ) 0.4μ1

 Reverse primer (ΙΟηΜ) 0.4μ1

ddH ₂ 0 7·2μ1

 Final volume 20μ1

 The primer sequences used here are:

 ITGB4BP: Forward primer: 5'-CCCAACAATACCACCGACCAGGA-3'

 Reverse primer: 5'-GCCCTCCCTGATTGCTGAAGACA-3' GAPDH: Forward primer: 5*-GGGGAAGGTGAAGGTCGGAGTC-3'

 Reverse primer: 5'-TCGCTCCTGGAAGATGGTGATG-3' Reaction conditions: denaturation at 95 °C for 2 min; denaturation at 95 °C for 15 seconds, annealing at 55 °C for 35 seconds, extension at 72 °C for 3 seconds, for a total of 40 cycles.

Signal acquisition was performed at 72 并 and analyzed using the 7500 system software of ABI, USA, and the results are shown in FIG. Example 6. Screening of ITGB4BP gene interference with effective lentivirus

 Because the constructed lentiviral interference vectors are not all interfering, they must be screened to select the most effective group for the experiment.

The screening was performed using a 24-well plate (Corning, USA). The number of scar fibroblasts per well was about 5×10 ³ , and the lentivirus 200 μ1 obtained in Example 4 was added to each well. Four days later, real-time PCR was performed on the cells. The technique detects the expression of mR A of ITGB4BP in the infected lentivirus-FrV-Hl/U6-copGFP-ITGB4BPRNAil#/2#/3# and the negative control infection group.

 The primer sequence used was (same as in Example 5):

 ITGB4BP: Forward bow I: 5'-CCCAACAATACCACCGACCAGGA-3'

 Reverse primer: 5'-GCCCTCCCTGATTGCTGAAGACA-3' GAPDH: Forward primer: 5'-GGGGAAGGTGAAGGTCGGAGTC-3'

 Reverse primer: 5'-TCGCTCCTGGAAGATGGTGATG-3' The specific steps are as follows: Observing the scar expression of scar fibroblasts after infection with lentivirus, the expression of the cells is about 50%, the cell morphology is long spindle-shaped, the negative control group has no fluorescence, and the medium is discarded; Tripure, RNA of fibroblasts was extracted according to the method of 5.1 in Example 5, and reverse transcription reaction was carried out using the extracted RNA as a template, and the reverse transcription primers, reaction system and reaction conditions were all reversed from 5.2 in Example 5. Record the same reaction. Then, using the obtained reverse transcription product as a template, real-time PCR detection was carried out in accordance with the method of 5.3 in Example 5, and the primers used were the same as those used in Example 5.3, and the reaction system and reaction conditions were also the same.

 The signal was collected at 72 °C and analyzed by 7500 system software. The results showed that in Figure 2A, the last interference-impaired lentivirus was lentivirus-FIV-Hl/U6-copGFP-ITGB4BPRNAi2# for subsequent experiments. . Example 1. Expression of fibrosis-related indicators after low expression of ITGB4BP in hypertrophic scar fibroblasts

According to previous studies, cells will secrete fibrosis-associated proteins such as TGF-pi, type I collagen, and MMP-9 in cell culture supernatants, and their expression changes can be understood by detecting the secreted expression of these proteins. Using the changes in the expression levels of these fibrosis-related proteins, you can push The formation of scars was measured.

 The expression of extracellular matrix proteins TGF-pi, type I collagen and MMP-9 in the supernatant of ITGB4BP in hypertrophic scar fibroblasts was inhibited by ELISA after mRNA level inhibition.

Briefly, it has been demonstrated in Example 6 that lentivirus-FIV-H1 /U6-copGFP-ITGB4BPRNAi2# is effective in inhibiting the expression of ITGB4BP mRNA in scar fibroblasts. The scar fibroblasts were passaged in a 24-well plate (Coming, USA) for experiments. The number of scar fibroblasts per well was about 5×10 ³ , and the lentivirus-FIV-Hl U6-packed according to the method of Example 4 was added to each well. copGFP-ITGB4BPRNAi2# 200μ1, added with 10°/. The calf serum was cultured in DMEM 800 μΐ, 37"C, 5% C0 ₂ incubator, and the cultured scar fibroblast supernatant was collected four days later. The supernatant was used to detect TGF-|31, type I collagen and ELISA. The content of MMP-9.

The experimental procedure for detecting TGF-βΙ by ELISA (refer to the instructions attached to the human TGF-βΙ ELISA test kit of American RB Company): Add ΙΟΟμΙ to the dilution (ie, serial dilution, serial dilution of 2000ng/ml high concentration standard) Standards of 1000 ng/ml, 500 ng/ml, 250 ng/ml, 125 ng/ml, 62.5 ng/ml, 31.25 ng/ml, 15.6 ng/ml, Ong/ml) were prepared in duplicate in the corresponding reaction plates; Add ΙΟΟμΙ samples (ie, experimental group: scar fibroblasts + ITGBRBP R Ai, and scar fibroblasts + negative control) in turn, 4 wells; gently mix for 30 seconds, seal the plate with plastic wrap, 37° C incubation for 60 min; wash the plate, drain the liquid in the plate, wash the reaction plate with washing solution (add 350 μl washing solution per well), remove water droplets (dry on thick stack of absorbent paper); wash 5 times repeatedly; The wells were added to ΙΟΟμΙ lx biotin (provided with the kit). Gently mix for 30 seconds, seal the plate well, incubate at 37 ° C for 60 min; wash the plate, drain the liquid in the plate, wash the reaction plate with washing solution (add 350 μl washing solution per well), and remove water droplets (in thick Dry on the stack of absorbent paper); Wash 5 times repeatedly; add ΙΟΟμΙ ΙχΗΙ 每 (with the kit) to each well. Gently mix for 30 seconds, seal the plate hole, incubate at 37 ° C for 30 min; wash the plate, drain the liquid in the plate, wash the reaction plate with washing solution (add 350 μl washing solution per well), and remove water droplets (in thick Repeat the washing on the stack of absorbent paper; 5 times; add ΙΟΟμΙ ΤΜΒ color solution to each well, mix gently for 10 seconds, incubate for 25 minutes at 37 °C in the dark (15 ± 10 minutes); Liquid (taken with the kit). Mix gently for 30 seconds; read the OD value at 450 nm using a 680 microplate reader for 30 minutes. The concentration of TGF-βΙ was determined according to the standard. It should be noted here that the US RB company's human TGF-βΙ test kit, human type I collagen test kit and human MMP-9 test kit can be used to detect different indicators in the same sample, specifically, in this experiment. , divide a sample into 3 equal parts a, b and c, a for TGF-βΙ detection kit to detect TGF-βΙ, b for type I collagen detection kit for detection of type I collagen, and c for MMP-9 The test kit detects MMP-9. The reagents used in the three tests are all carried out in the kit, and the detection steps are the same as those described above for detecting TGF-βΙ using the human TGF-βΙ detection kit. Example 8. Expression of fibrosis-related indicators after high expression of ITGB4BP in normal fibroblasts

The experiment was carried out using a 24-well plate, and the number of normal skin fibroblasts per well was about 5×10 ³ , and each of the adenovirus Ad-EGFP and Ad-ITGB4BP-EGFP obtained in Example 3 was added 200 μl each, and 10% was added. The calf serum was cultured in DMEM 800 l, 37 ° C, 5% C0 ₂ incubator, and the content of TGF-pi, type I collagen and MMP-9 in the supernatant was measured by ELISA five days later (method is the same as in Example 7, The human TGF-β Ι detection kit of the US RB company, the human type I collagen detection kit, and the human MMP-9 detection kit were used, respectively. Example 9. Expression of fibrosis-related indicators after high expression of ITGB4BP in hypertrophic scar fibroblasts

 9.1 mRNA level detection a-SMA, TGF-βΙ and type I collagen expression:

The experiment was performed using a six-well plate, and the number of hypertrophic scar fibroblasts per well was about 8χ10 ⁴ , and each of the adenovirus Ad-EGFP and Ad-ITGB4BP-EGFP obtained in Example 3 was added to each of 600 μl, and the addition was 10 % calf serum was cultured in DMEM 2400 l, 37 ° C, 5% C0 ₂ incubator, and the expression of a-SMA, TGF-βΙ and type I collagen was detected by real-time fluorescent quantitative PCR after five days (RA extraction, The experimental method of reverse transcription reaction and real-time fluorescent quantitative PCR is the same as in Example 5).

 The primer sequences used are:

TGF-β 1: forward primer: 5'-TGGAAACCCACAACGAAATCTATGA-3' reverse primer: 5'-TGGAAACCCACAACGAAATCTATGA-3' type I collagen: forward primer: 5'-tcccaccaatcacctgcgtaca-3' Reverse primer: 5'-cgccggtggtttcttggtcg-3'

 α-SMA: positive bow I: 5'-cggctttgctggggacgat-3'

 Reverse primer: 5 '-caggggcaacacgaagctcat-3 '

9.2 Protein level detection TGF-pi, and α-SMA expression:

The experiment was carried out using a six-well plate, and the number of cells per well was about 8 χ 10 ⁴ , and each of the adenovirus Ad-EGFP and Ad-ITGB4BP-EGFP obtained in Example 3 was added to 600 μl each, and 10% fetal calf serum was added thereto. The cells were cultured in DMEM 2400 1, 37 ° C, 5% C0 ₂ incubator, and the expression of TGF-βΙ and a-SMA in the cells was detected by Western blotting five days later.

9.2.1 Extraction of proliferative scar fibroblast protein (RIPA method):

The specific steps are as follows: Treat the cells on ice, wash the cells with pre-cooled PBS, add 200μ1 RIPA cell lysis (containing 1% PMSF) solution per well, repeatedly scrape the cells with ice on the cell curette, and transfer to a clean 1.5ml. EP tube, placed on ice for 30 min; ultrasonic cracking in ice bath, 4. C 14000 rpm x 30 min, the supernatant after centrifugation was transferred to a new 1.5 ml EP tube; the protein concentration was determined by BCA method (refer to the US Pierce BCA Protein Concentration Kit). Adjust the concentration of the sample to the appropriate concentration with ddH ₂ 0, add 5XSDS-PAGE loading buffer; boil the protein for 5 min; load 20 μl/well; SDS-PAGE electrophoresis, electrophoresis, remove the gel and put it into the electroporation buffer Soak for 20min, soak 6 filter papers at the same time, 1 PVDF film, install "sandwich" structure, from negative electrode to positive electrode: three-layer filter paper-gel-PVDF film-three-layer filter paper, constant current 0.8Ma/cm ² , electric rotation 1.5 hours . Remove PVDF and block in TBST (1%. Tween20) blocking solution containing 5% skim milk powder for 2 hours at room temperature; add primary antibody (mouse anti-human β-actin monoclonal antibody 1. 400, mouse anti-human TGF -βΙ monoclonal antibody 4 g/ml, mouse anti-human α-SMA monoclonal antibody 1: 100 ) overnight incubation at 4°C; washing membrane l%oTBSTx l0minx 3 times; addition of secondary antibody (peroxidase-labeled goat antibody) Mouse IgG, 1: 2000) Incubate for 1 hour at room temperature; wash membrane 1%. TBSTx l0minx3 times; developed, added to the Santa Cruz company's chemiluminescence solution A liquid B solution (both included in the kit), each closed after 500μ1 development.

 SDS-PAGE electrophoresis gel system:

 Separation gel (12%), 80V electrophoresis Total volume 5ml Laminated gel (5 %), 150V electrophoresis Total volume 2ml

30% acrylamide 2ml 30% acrylamide 0.33ml

1.5mol/L Tris-HCL 1.3ml 1.5mol/L Tris-HCL 1.3ml

10% SDS 0.05ml 10% SDS 0.02ml

10% persulfate hinge 0.05ml 10% ammonium persulfate 0.02ml

TEMED 0.002ml TEMED 0.002ml ddH ₂ 0 1.6ml ddH ₂ 0 1.4ml 9.3 Using FPCL model to observe the effect of ITGB4BP on the biological behavior of fibroblasts

Preparation of FPCL model: Hypertrophic scar fibroblasts were infected with adenovirus Ad-EGFP and Ad-ITGB4BP-EGFP for 5 days, and then resuspended in serum-free DMEM with 0.25% trypsin to prepare a cell suspension. And adjust the cell density to lx lO ⁶ cells / ml. According to the cell suspension: 5xDMEM: rat tail collagen = 1: 2: 7 volume ratio mix the above various components, specifically first mix the rat tail collagen and 5xDMEM on ice, adjust the pH 7.2, then add the cell suspension; The box was placed for 30 min to form a gel, and then cultured by adding 3 ml of serum-free DMEM to each well. That is, a fibroblast-populated collagen lattice (FPCL) is obtained.

 3 FPCL for each group. On the 8th day, the diameter change of the FPCL was observed and recorded, and the shrinkage index was calculated. The formula for calculating the FPCL shrinkage index is as follows:

CI=[1-(D/D ₀ ) ² ] xl 00%

 Where: CI: contraction index; D: diameter of collagen gel block; Do: initial diameter of gel block (22 mm).

 In addition, it should be noted that in all embodiments of the present invention, if statistical analysis is required, the data is subjected to independent sample testing using SPSS 13.0 software, and Ρ ≤ 0.05 is considered to have statistical significance. Results and discussion

 1. ITGB4BP is underexpressed in hypertrophic scar fibroblasts

 The test results of Example 5 are shown in Fig. 1. Figure 1 shows that ITGB4BP can be expressed in normal skin and hypertrophic scar fibroblasts, which represents the raw relative quantitation (hereinafter referred to as raw RQ) values of mRNA expression levels of 1 and 0.704, respectively, indicating that ITGB4BP is proliferative. Low expression in scar fibroblasts. This suggests that low expression of ITGB4BP may be associated with hyperfibrosis in hypertrophic scars, ie low expression of ITGB4BP exacerbates fibrosis of hypertrophic scars.

 2. In hypertrophic scar fibroblasts, inhibiting the expression of ITGB4BP can aggravate the fibrosis of scars

Figure 2A corresponds to the results of Example 6 and shows: lentivirus ^ -111/1; 6 (^0??-1 Ding 0848?1^8 〖2# at the mRNA level interferes with ITGB4BP The best. Specifically, the negative control group at the mRNA level was 1.00 original RQ, and the expression level of 1# lentiviral vector for ITGB4BP was 2.068 original RQ, which had no interference effect on ITGB4BP; the expression of 2# vector to ITGB4BP was 0.025 original RQ, inhibition rate 99%, that is, the most interference effect; 3# vector to ITGB4BP expression is 0.253 original RQ, the inhibition rate is about 75%. Figure 2B corresponds to the results of Example 7 and shows that TGF-β MMP9 and type I collagen are expressed in hypertrophic scar fibroblasts after inhibiting the expression of ITGB4BP at the mRNA level in hypertrophic scar fibroblasts. Increased. Compared with the negative control, TGF-βΙ increased about 5 times, MMP9 increased about 4.5 times, and type I collagen increased about 6 times, indicating that at the mRNA level, ITGB4BP can significantly increase fibrosis-related indicators after inhibition. For example, the expression of TGF-pi, MMP9 and type I collagen significantly aggravated fibrosis.

 3. In normal skin fibroblasts, ITGB4BP expression was increased to inhibit fibrosis. The results of Example 8 are shown in Fig. 3. The results showed that TGF-β MMP9 and type I collagen were down-regulated in normal skin fibroblasts after elevated expression of ITGB4BP mRNA in normal skin fibroblasts. Specifically, TGF-βΙ decreased by about 2 times, MMP9 decreased by about 4.5 times, and type I collagen decreased by about 6 times. That is, high expression of ITGB4BP can significantly inhibit the expression of fibrosis-related indicators such as TGF-β MMP9 and type I collagen. Inhibition of fibrosis.

 The specific data corresponding to Figure 2 and Figure 3 are as follows:

Table 2.1 TGF-βΙ standard concentration and corresponding OD ₄₅₍₎ detection standard (ng/ml) 1000 500 250 125 62.5 31.25 15.625 0

 Group 1 0.540 0.478 0.299 0.207 0.149 0.108 0.093 0.076

OD ₄₅₀

Group 2 0.577 0.502 0.328 0.225 0.146 0.117 0.098 0.092 Regression analysis based on TGF-βΙ standard concentration (X) and its corresponding OD ₄₅₍ ) value (y) (Table 2.1), using a quadratic curve model to fit the correlation The coefficient (R) value is 1.000, the F value in the analysis of variance is 3464.810, and the significance probability SignifF=0.000<0.05, indicating that there is a highly significant quadratic function relationship between the two variables, and the fitted quadratic curve equation is y. = 0.079 + 0.001x - 0.00000066X ² , and a standard curve of TGF-β Ι concentration and OD ₄₅₀ value is obtained accordingly. Table 2.2 OD ₄₅ o detection values of fibroblasts producing TGF-βΙ under different stimulation conditions (detection by human TGF-P1 ELISA kit)

Hypertrophic scar fibroblast normal skin fibroblast Negative control ITGB4BP RNAi EGFP ITGB4BP-EGFP Group 1 0.114 0.359 0.447 0.247

 Group 2 0.151 0.451 0.472 0.377

OD ₄₅₀

 Group 3 0.150 0.449 0.519 0.253

Group 4 0.154 0.334 0.496 0.374 Table 3.1 Standard concentration of MMP-9 and corresponding OD _45Q test value standard (ng/ml) 1000 500 250 125 62.5 31.25 15.625 0

 o

0.094 根据 MMMP-9得到的标准品浓度 (x)和对应的 OD₄₅o值 (y)进行回归分析 (表 3)， 选 用三次曲线模型拟合得到的相关系数 (R)值为 0.996, 方差分析中 F值 =154.414， 显著 性概率 SignifF=0.000<0.05，说明这两个变量之间存在高度显著的三次函数关系，其三 次曲线回归方程式为 y=0.106+0.003x_0.0000067x²+0.00000000399x³， 据此得 MMP-9 浓度与 OD₄₅()值的标准曲线。 表 3.2 成纤维细胞在不同刺激条件下生成 MMP-9的 OD_45Q检测值（人 MMP-9 ELISA 检测试剂盒检测） 0.088

0.094 According to the standard concentration (x) obtained by MMMP-9 and the corresponding OD ₄₅ o value (y) for regression analysis (Table 3), the correlation coefficient (R) value obtained by fitting the cubic curve model is 0.996, analysis of variance The medium F value = 154.414, the significance probability SignifF = 0.000 < 0.05, indicating that there is a highly significant cubic function relationship between the two variables, and the cubic curve regression equation is y=0.106+0.003x_0.0000067x ² +0.00000000399x ³ , Based on this, a standard curve of MMP-9 concentration and OD ₄₅ () value is obtained. Table 3.2 OD _45Q detection values of fibroblasts producing MMP-9 under different stimulation conditions (detection by human MMP-9 ELISA kit)

 Hypertrophic scar fibroblasts normal skin fibroblasts

 Experimental grouping

 Negative control ITGB4BP RNAi EGFP ITGB4BP-EGFP Group 1 0.199 0.579 0.830 0.528

 Group 2 0.213 0.832 0.481

OD ₄₅₀

 Group 3 0.204 0.749 0.527

Group 4 0.219 0.554 0.827 0.524 Table 4.1 Concentration of type I collagen standard and corresponding OD _{45 ()} test value standard (ng / ml) 1000 500 250 125 62.5 31.25 15.625 0 Group 1 0.475 0.427 0.370 0.278 0.215 0.132 0.107 0.090

OD ₄₅₀

Group 2 0.523 0.488 0.367 0.262 0.186 0.138 0.101 0.082 According to the type I collagen standard concentration (x) and the corresponding OD4 _5Q value (y) for regression analysis (Table 4.1), the correlation coefficient (R) value obtained by fitting the cubic curve model is 0.999, and the F value in the variance analysis = 577.326, Significant probability Signif F=0.000<0.05, the fitted cubic regression equation is y=0.086+0.002x - 0.0000028x ² +0.00000000139x ³ , and the standard of type I collagen concentration and OD ₄₅ o value is obtained accordingly. curve. Table 4.2 OD ₄₅₍₎ detection value of fibroblasts producing type I collagen under different stimulation conditions (human type I collagen

 ELISA test kit detection)

 Hypertrophic scar fibroblasts normal skin fibroblasts

 Experimental grouping

 Negative control ITGB4BP R Ai EGFP ITGB4BP-EGFP Group 1 0.144 0.327 0.632 0.416

 Group 2 0.123 0.323 0.504 0.381

OD ₄₅₀

 Group 3 0.146 0.321 0.798 0.421

 Group 4 0.145 0.320 0.653 0.429

4. In hypertrophic scar fibroblasts, high expression of ITGB4BP has significant anti-fibrosis effect.

 The experimental results of Example 9 are shown in Figures 4-7. The results in Figure 4 indicate that in hypertrophic scar fibroblasts, increased expression of ITGB4BP inhibits the expression of myofibrillar surface marker α-SMA, gp, which inhibits the phenotype of fibroblasts to myofibroblasts. Conversion. After high expression of ITGB4BP in hypertrophic scar fibroblasts, the expression level of a-SMA mRNA was 0.329 original RQ, which was lower than the 1.00 original RQ of the control group, ie, the inhibition rate of a-SMA was 67% (Fig. 4A). The results of Western blotting experiments in Figure 4B also clearly showed that the high expression of ITGB4BP inhibited the expression of a-SMA at the protein level, and further quantified that the relative expression level of a-SMA protein in the high expression group of ITGB4BP was 0.016, which was lower than that of the control group 0.023. The inhibition rate was 30% (Fig. 4C).

The results in Figure 5 indicate that elevated expression of ITGB4BP inhibits the expression of TGF-βΙ in hypertrophic scar fibroblasts in hypertrophic scar fibroblasts (or models). After high expression of ITGB4BP in hypertrophic scar fibroblasts, the expression level of TGF-βΙ mRNA was 0.3979 original RQ, which was lower than the 1.00 original RQ of the control group, that is, the inhibition rate of TGF-βΙ was 60%. The results of Western blotting experiments in Figure 5B also clearly indicate that high expression of ITGB4BP is in the protein. The expression of TGF-βΙ was inhibited at the level, and further quantified showed that the relative expression level of TGF-βΙ protein in the high expression group of ITGB4BP was 0.008, which was lower than that of the control group 0.069, and the inhibition rate was 88% (Fig. 5C).

 The results in Figure 6 indicate that elevated expression of ITGB4BP inhibits the expression of type I collagen in hypertrophic scar fibroblasts in hypertrophic scar fibroblasts (or models). After high expression of ITGB4BP in hypertrophic scar fibroblasts, the expression level of type I collagen mRNA is

0.480 . Starting RQ, which is lower than the 1.00 original RQ of the control group, that is, the inhibition rate of type I collagen is 52.

%.

 The results in Figure 7 indicate that ITGB4BP inhibits contraction of myofibroblasts in hypertrophic scar fibroblasts (or models). After high expression of ITGB4BP in hypertrophic scar fibroblasts, the contraction index of the three-dimensional gel was 0.23, and that of the control group was 0.90, that is, the contractility of the gel decreased by about 74%. From the above results, it can be seen that in fibrotic diseases, the low expression of ITGB4BP may be an important initiating factor regulating the phenotypic transformation and functional changes of fibroblasts. ITGB4BP may be a strongly involved fibrosis formation. Negative signal. Therefore, the high expression of ITGB4BP has obvious anti-fibrotic effect. Therefore, ITGB4BP and its therapeutic gene expression vector can be used to prevent and/or treat hypertrophic scar disease or fibrotic lesions.

 It will be understood that the invention has been particularly shown and described with reference to the exemplary embodiments thereof The spirit and scope of the invention as defined by the claims. references:

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Claims

Rights request 1. β4 integrin-binding protein (ITGB4BP) (SEQ ID NO 1 ) and its derivatives, or recombinant gene expression vector expressing ITGB4BP and its derivatives are prepared for the prevention and/or treatment of hypertrophic scar or fibrotic lesions. Use in pharmaceutical compositions or kits.  2. The use of claim 1, wherein the ITGB4BP derivative is a protein derivative that retains a binding site and a functional site of ITGB4BP (SEQ ID NO 1), including retention inhibition Expression of a fibrosis-related index such as TGF-βΚ MMP9 or type I collagen, or a protein derivative that inhibits the expression of a fibroblast surface marker (x-SMA expression).  3. The use of claim 1 or 2, wherein the hypertrophic scar comprises skin damage such as burns, burns, chemical damage, freezing injuries, cut injuries, and the like.  The use according to claim 1 or 2, wherein said fibrotic lesions include pathological fibrosis of various tissues and organs, including skin tissue fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis and the like.  5. The use of claim 4, wherein said fibrotic lesions comprise a variety of pathological contractions caused by fibrosis, including body surface and internal organs, pathological contractions of tissues, and the like.  The use of claim 1, wherein the expression of TGF-pi, MMP9 and type I collagen in the fibrosis-related indicators is significantly decreased after the high expression of the ITGB4BP in normal skin fibroblasts, and fibrosis is inhibited.  7. The use according to claim 6, wherein after the high expression of the ITGB4BP in hypertrophic scar fibroblasts, the expression of the myofibrillar surface marker α-SMA is decreased, and the hypertrophic scar fibroblasts are inhibited to the myofibroblasts. Transformation, and inhibition of contractility of myofibroblasts.  A pharmaceutical composition or kit for preventing and/or treating a hypertrophic scar disease or a fibrotic lesion, the pharmaceutical composition or kit comprising a therapeutically effective amount of ITGB4BP (SEQ ID NO 1 ) or a derivative thereof, Or a recombinant gene expression vector expressing ITGB4BP or a derivative thereof, and a pharmaceutically acceptable excipient or carrier. 9. The pharmaceutical composition or kit of claim 8, further comprising a therapeutically effective amount of another scar or fibrotic lesion inhibitor. 10. A method of preventing and/or treating a hypertrophic scar or fibrotic lesion, the method comprising administering to a subject a therapeutically effective amount of ITGB4BP or a derivative thereof, or a recombinant gene expression vector expressing ITGB4BP or a derivative thereof, or The pharmaceutical composition or kit of claim 8.