CN1408431A - Gene engineering medicine for curing diseases relative to vascularization - Google Patents
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Abstract
The present invention relates to a kind of medicine containing human recombinant plasminogen K5 segment and for treating diseases relative to vascularization. Recombinant K5 is used in the experiment of human eye retina capillary endothelial cell growth inhibition, the in vivo experiment of preventing and inhibiting rat retina nerve vascularization and the experiment of K5 antitumor. The results show that recombinant K5 has obvious in vivo and in vitro inhibition on retina vascularization and can inhibit tumor growth obviously while has no poison and damage to non-vascular cell.
Description
Technical field:
The present invention relates to a kind of genetically engineered drug, particularly relate to and contain the medicine that people's recombinant plasminogen K5 fragment is used for the treatment of the disease relevant with the new vessels generation.
Background technology:
Angiogenesis is the result or the factor of a complex physical and pathological process, remove normal physiological condition (as wound healing, conceived and grow) can take place under the condition, under pathological conditions, congested all relevant with the generation of new vessels with the tumor generation as the retina that causes by diabetes.Especially retina neural vascularization, the blood capillary abnormal formation of neovascularity from pre-existing is the distinctive pathological phenomenon of many oculopathy.As diabetic retinopathy, meniscocyte's retinopathy, retinal vessel occlusion and retina precocity (ROP).Tumor, cerebral ischemia and chronic inflammatory disease also can cause the generation of new vessels, thereby increase the weight of these state of an illness.The angiogenesis ability of medical research proof solid tumor is vigorous especially, thereby fights for nutrition with normal body.If can suppress the generation of new vessels, just can effectively suppress tumor growth, and cause tumor mortality.The angiogenesis factor inhibitor is the gene of finding in recent years, and the protein product of this gene can suppress the formation of new vessels, comprising Angiostatin (plasminogen).Think that at present Angiostatin is formed through specific protease hydrolysis by the fiber proenzyme.Angiostatin comprises four kringle structures (ring structure that comprises three disulfide bond), and wherein independent kringle 1,2,3 also has the effect of angiogenesis inhibitor.Because the Angiostatin molecule is bigger, is not suitable as medicine.
Studies show that in recent years, K5 district (the kringle5 that plasminogen (Plasminogen) is inner, be designated hereinafter simply as K5, be present in Plasminogen, but do not belong to Angiostatin) propagation of endotheliocyte and migration there are stronger inhibitory action than Angiostatin.(Ji，W.R.，Barrientos，L.G.，Trail，P.A.，1998，BBRC?247，414-419?Cao，Y.，Chen，A.，Llinas，M.，1997，J.Biol.Chem.272，22924-22928)。
K5 be the proteoclastic fragment of a kind of plasminogen (plasminogen) (Cao, Y., Ji, R.W., Davidson, D., Folkman, J., 1 996, J.Biol.Chem.271:29461), be the 5th kringle district in the fiber proenzyme.Each kringle forms (Castellino FJ, McCanceSG:The kringle domains of human plasminogen.Ciba Foundation Symposium212:46-65,1997) by 80 aminoacid.Verified, the K5 inhibition of endothelial cell proliferation; And it suppresses activity and is higher than angiostatin (being kringle 1-4) (Cao, Y., Ji, R.W., Davidson, D., Folkman, J., 1996, J.Biol.Chem.271:29461, Cao, Y., Chen, A., Llinas, M., 1997, J.Biol.Chem.272,22924-22928, O ' Reilly, M.S., Holmgren, L., Shing, Y., Folkman, J., 1994, Cell.79:315).In addition, K5 also suppresses the important step (Ji of this angiogenesis of endothelial cell migration, W.R., Barrientos, L.G., Trail, P.A., 1998, BBRC 247,414-419, Lu H, Dhanabal M, Volk R, Waterman MJF, Ramchandran R, Knebelmann B, Segal M, Sukhatme VP:Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells.BiochemBiophy Res Comm 285:668-673,1999).K5 to endotheliocyte effect work by inhibition of inducing cell loop cycle and apoptosis, and be directed to the propagation of endotheliocyte.Because that it has is efficient, the selectivity of cell type, and short aminoacid sequence, K5 is considered to treat the active substance of neovascular disease.
But as medicine, be used for the treatment of tumor, the retinal diseases relevant and do not appear in the newspapers as yet with the new vessels generation with Plasminogen K5.
Summary of the invention:
The objective of the invention is to develop the segmental medicine of a kind of Plasminogen of containing K5, be used for the treatment of and relevant tumor and the retinal diseases of new vessels generation.
The present invention has found that reorganization K5 has obvious inhibitory action with external to retina and tumor neovascularization in vivo.
Technical scheme of the present invention is:
1.K5 acquisition
1) makes up people's K5 expression system of recombinating
Utilize the RT-PCR technology, from people's liver rna, amplify K5cDNA, on the Eco RI and the site of HindIII of this PCR product cloning in the pET22 carrier, formed the fusion gene sequence of 6X histidine and K5.The correctness of expression vector establishment is confirmed by order-checking.
2) people's expression and purification of K5 of recombinating
With above-mentioned carrier Transformed E .coli bacterial strain BL-21/DE3, the people that this bacterial strain is expressed recombinates K5 mainly in plasmalemma (preplasmic) gap.Recombinant Protein Expression is inducing by IPTG.Reclaim the albumen in plasmalemma gap with lysozyme and centrifugation method.The albumen that obtains is thus carried out separation and purification.The people who the obtains K5 albumen of recombinating is confirmed with SDS-PAGE method and Western blotting.
2. pharmacodynamic experiment
Carried out following biological experiment with medicine of the present invention:
1) reorganization K5 tests human eye retina's capillary endothelial cells (HRCEC) growth inhibited
The result shows: HRCEC is at hypoxia (1%O
2) under the condition than quantity showed increased under normal oxygen condition (p＜0.05, n=4), its half-inhibition concentration EC
50Under normal oxygen and hypoxia condition is respectively 70nM and 600nM.
2) the cellular type specificity of K5 suppresses:
The result shows: K5 to former send out theca cell (Pericyte) all or E1A-NR3 cell (from the deutero-a kind of immortalized cell of rat retina neuron system) unrestraint effect (p＞0.05, n=4).The inhibitory action of proof K5 only is directed to neural endotheliocyte.
3) experiment in the K5 prevention rat retina neural blood vessel organizator
The result shows: the animal eye that K5 handled obviously reduces (K5 experimental group 33.3 ± 2.7nuclei/section than matched group retina neural blood vessel precursor; PBS matched group 66.1 ± 5.9, P＜0.01, n=8).Proof injection K5 can prevent the development of retina neural blood vessel.And the inhibitory action of prompting K5 depends on concentration.
4) K5 suppresses the interior experiment of body of retina neural angiogenic growth
Behind the retina neural angiogenesis, with the eyes that K5 handles, the average of its retina neural angiogenic growth only has marginal increase, (inject preceding 74.6 ± 12.4nuclei/section, injection back 80.3 ± 22.5P=0.4, n=8).The average of the matched group retina neural angiogenic growth of mutually anticaustic PBS has increased by 1 times and (has injected preceding 74.6 ± 12.4nuclei/section, inject 145.5 ± 24.1 nuclei/section P＜0.01, back, n=8).Yet K5 does not reduce the quantity of vascular cell precursor.This results suggest can suppress neurovascular growth after the K5 shot, and not influence the vascular endothelial cell precursor.
5) safety experiment of K5
The infiltration of no tangible Histological change or inflammatory cell takes place behind the ocular injection K5.In addition, blood vessel can not detectedly change after injection in the normal retina.This discovery prompting: K5 can not cause retina or normal blood vessels toxicity under used concentration.
6) antitumor action of K5
Personnel selection reorganization K5 albumen carries out therapeutic test to the rat of retina hyperemia and the nude mice of generation tumor respectively.To the rat of retina hyperemia, have the nude mice of tumor, carry out the in-situ injection of tumor with K5, after 4 weeks, take off tumor, carry out the lump size and analyze and morphologic analysis.Experiment shows that K5 can suppress growth of tumor significantly.
Advantage of the present invention and effect
1) gene outcome of the used people K5 of the present invention is the human body endogenous protein.Utilize bacterial fermentation production people K5 gene outcome, belong to biological high-tech, pollute little, the productive rate height, cost is low.
2) people's K5 polypeptide of recombinating is 16kD, and is all littler, preferably as drug molecule than the molecular weight of the angiogenesis factor inhibitor of other natural human protide.
3) medicine of the present invention all can effectively suppress vascular cell growth in external body, and this kind inhibition is specific, non-vascular cell is not produced and suppresses.Promptly non-vascular cell is not produced and poison and damage.
The specific embodiment:
The preparation of embodiment 1 K5
1. make up people's K5 expression system of recombinating
Utilize RT-PCR (inverse transcription polymerase chain reaction) technology, from people's liver rna, amplify K5cDNA, on the Eco RI and the site of Hind III of this PCR product cloning in the pET22 carrier (U.S. Novagen company product), formed the fusion gene sequence of 6X histidine and K5.The correctness of expression vector establishment is confirmed by order-checking.
2. people's expression and purification of K5 of recombinating
With above-mentioned carrier Transformed E .coli bacterial strain BL-21/DE3 (U.S. Novagen company product).The people that this bacterial strain is expressed recombinates K5 mainly in plasmalemma (preplasmic) gap.Recombinant Protein Expression is inducing by IPTG.Reclaim the albumen in plasmalemma gap with lysozyme and centrifugation method.The albumen that obtains is thus crossed His-Bind post (U.S. Novagen company product) carry out separation and purification.The people who obtains behind the purification K5 albumen of recombinating is confirmed with SDS-PAGE method and Western blotting.
Embodiment 2 K5 are to the human retina cell growth inhibition test
1. experiment material
Retina cell source: obtain volunteer's human eye from association of American South card Lion eye bank (theSouth Carolina Lion ' s EyeBank Association).
2. experimental technique
A. separating and cultivation of human eye retina's capillary endothelial cells (HRCEC) and all theca cells (Pericyte):
From human eye, isolate retinal capillary endothelium cell (HRCEC) and mix that (DiI 1 with fluorescent probe labelling acetylation low density lipoprotein, LDL; 1 '-dioctadecyl-3; 3; 3 '; 3 '-tetramethylindocarbocyanineperchlorate, Biomedical Technologies Inc., Stoughton; MA), determine the purity of second filial generation endotheliocyte in the culture.Document (Grant MB is pressed in the separation of week theca cell (Pericyte), Guay, C:Plasminogen activator production by human retinal endothelial cells ofnon-diabetic and diabetic origin.Invest Ophthalmol Vis Sci 32:53-64,1991) method is carried out.
B. viable count:
To be in second and hexabasic above-mentioned cell inoculation in being coated with 12 well culture plates of gelatin/fibronectin, the cell inoculation density in every hole is 0.8 * 104, in containing the culture medium of 15% FBS, cultivate after 24 hours, change to containing the culture medium of 5%FBS and 1ng/ml bEGF, add the K5 of variable concentrations in the culture medium of changing, every kind of concentration is established 4 repetitions.Then, cell is at normal oxygen concentratio and low oxygen concentration (1%O
2) condition under cultivated respectively 72 hours.By the trypsinized method, from culture plate, take out cell, be resuspended in the somatomedin (growth media), with 0.4% trypan blue (trypan blue) dyeing, under optical microscope, carry out viable count then.Analyze the average cell number with Student ' s test, with the inhibition of definite K5 cell growth and the specificity of inhibition.
3. experimental result:
Reorganization K5 under normal oxygen and hypoxia condition to the body outer suppressioning experiment of HRCEC
With concentration be 20,40,80, the reorganization K5 of 160nM handles HRCEC, cultivates respectively 72 hours under normal oxygen and hypoxia condition, carries out cell counting then.When concentration during at 20nM, K5 just can obviously reduce cell quantity (p＜0.01 n=4) under normal oxygen and hypoxia condition.Half-inhibition concentration EC
50Under normal oxygen and hypoxia condition is respectively 70nM and 600nM.
Conclusion: HRCEC is at hypoxia (1%O
2) under the condition than quantity showed increased under normal oxygen condition (p＜0.05, n=4).
The cellular type specificity of embodiment 3 K5 suppresses
Experiment condition is with embodiment 2, the result show K5 do not show any significant to former send out theca cell (Pericyte) all or the inhibitory action of E1A-NR3 cell (from the deutero-a kind of immortalized cell of rat retina neuron system) (p＞0.05, n=4).The concentration of K5 all can be observed this from 20-320nM.Prompting: the inhibitory action of K5 only is directed to neural endotheliocyte.
Retina neural is angiopoietic in embodiment 4 animal bodies induces
1. laboratory animal: buy Spragre Dawley and BrownNorway rat from the Harlan that is positioned at Indianapolis.
2. experimental technique:
Angiopoietic the inducing with quantity of retina neural determined by document (Smith LE, et al:Oxygen-induced retinopathy in the muse.Invest Ophthalmol Vis Sci35:101-111.1994, Smith LEH et al:Essential role of growth hormone inischemia-induced retinal neovascularization.Science 276:1706-1709,1997) method is carried out, and change is arranged slightly.With 8 albinic Spragre Dawley and Brown Norway neonate rat random packet.The back 7 days rat that is born is exposed to (75%O under the low-oxygen environment
2) 5 days, under normal oxygen condition, raised 7 days then, to induce the retina neural vascularization.
3. the angiopoietic check of retina neural
Western blotting is analyzed: with the retina section, carry out the ultrasonotomography homogenize then.Protein concentration with BioRad albumen test determination supernatant.50 microgram soluble proteins are through SDS-PAGE (15% polyacrylamide gel) electrophoresis, and electrotransfer is to Hybond ECL nitrocellulose filter membrane (AmershamInternational plc).Use 5%BLOTTO TBST buffer (137mMNaCl, 0.1%Tween 20 for 20mM Tris-Cl, pH7.6) solution jolting at room temperature 1 hour, to block this film then.(Santa Cruz, CA) to this bovine lacto transfer technique optimizer, concentration is 1: 4000, cultivates jolting with this film at 4 ℃ and spends the night to add the rabbit anti-VEGF antibodies.Under the room temperature this film is washed three times each 20 minutes with TBST.The anti-rabbit antibody of donkey with horseradish peroxidase (Amersham International plc) labelling claims to release to 1: 4000 with bovine lacto transfer technique optimizer solution, at room temperature with this film incubation 2 hours.This film with TBST flushing three times, is detected box (Amersham International plc) with ECL this film is developed the color into band, use the KodakX-Omat exposure then.Same film carries out band shapeization and Western blot once more with being specific to the b-actin antibody.
The Western blotting analysis confirms in the inductive Brown Norway of oxygen rat retina pathological changes, the generation quantity of retina angiogenesis cell is more than Spragre Dawley rat before the Brown Norway rat, and therefore Brown Norway rat is all adopted in experiment subsequently.The inductive Brown Norway of oxygen rat retina pathological changes: in all experimental rats, can be observed tangible retina neural vascularization, microaneurysm, non-perfusion area and bleeding.
Experiment in the embodiment 5 K5 prevention rat retina neural blood vessel organizator
Laboratory animal is originated and is induced the pathological model method with embodiment 4.
Experimental technique:
People's K5 albumen of recombinating is dissolved in PBS, and 1mg/ml carries out filtration sterilization.Part animal is injected K5 immediately change normal oxygen environment over to from low-oxygen environment after, experiment showed, this moment, new neural blood vessel does not take place as yet form.After raising 7 under the normal oxygen environment, check retina neural.
Another part animal is injected K5 after 7 days changing normal oxygen environment over to from low-oxygen environment, animal after the injection was raised 7 days under normal oxygen condition,, variable concentrations K5 is expelled in the vitreous body of right eye with the capillary glass tube pin with laboratory animal anesthesia, left eye is injected PBS in contrast.Animal after the injection is killed Mus after raising 7 days under the normal oxygen condition, take out the eye body, use 10% formalin fixed, do section.With hematoxylin and eosin dyeing.Count with the vascular cell nuclear of 250 times of optical microscopes, and 8 sagittate sections of every eye are carried out morphocytology detect retina glass side.The vascular cell check figure order of every treated animal averages.This average and matched group are compared, analyze with Student ' s test method.
The retinal vessel growing state method of inspection:
High molecular fluorescein visualization:
Press document (Smith LE, et al:Oxygen-induced retinopathy in the muse.InvestOphthalmol Vis Sci 35:101-111,1994) method, with laboratory animal anesthesia and pour into the fluorescein (50mg/ml that produces via injection Sigma in the ventricle, 2 * 106 molecular weight isothiocyanate glucosan fluorescein fluorescein isothiocyanate-dextran) after, put to death animal immediately, take out the eye body, fix 10 minutes with 4% paraformaldehyde, retina is done section, remove crystalline lens and vitreous body, in 4% paraformaldehyde, cultivated 3 hours.Retina after handling is tiled on the slide of a gelatin covering.(Axioplan2 Imaging Zeiss) checks vascular system down at fluorescence microscope.
Experimental result: accepted the eye of 3ul K5 (3ug/ul) K5 injection,, demonstrated the neural blood vessel clump and reduce compared with the matched group of using with volume PBS injection.The eye that K5 handled also obviously reduces (K5 experimental group 33.3 ± 2.7 nuclei/section than matched group retina neural blood vessel precursor; PBS matched group 66.1 ± 5.9, P＜0.01, n=8).
The result shows: disposable injection K5, but at least partial prophylaxis under the ischemia condition, the development of retina neural blood vessel.And the injection with volumetric concentration only for 0.3ug/ul K5 can not produce significant other neurovascular cells minimizing (65.3 ± 2.0 nuclei/section, P=0.9, n=8), the prompting K5 inhibitory action depend on concentration.
Embodiment 6 K5 suppress the interior experiment of body of retina neural angiogenic growth
Behind the retina neural angiogenesis, be to determine the effect of K5, after changing normal oxygen condition over to, K5 was injected in the rat vitreous body in 7, tangible retina neural angiogenesis has taken place this moment.Under normal oxygen condition, raising again 7 then, allowing the continued growth of newborn retina neural blood vessel, counting then.With the eyes that K5 handles, the average of retina neural angiogenic growth only has marginal increase, (inject preceding 74.6 ± 12.4nuclei/section, injection back 80.3 ± 22.5 P=0.4, n=8).The average of the matched group retina neural angiogenic growth of mutually anticaustic PBS has increased by 1 times and (has injected preceding 74.6 ± 12.4nuclei/section, inject back 145.5 ± 24.1nuclei/section P＜0.01, n=8).Statistical analysis shows, through eye that K5 handled compared with handled with PBS inject 7 after other neurovascular cells few (P＜0.05, n=8).Yet K5 does not reduce the quantity of vascular cell precursor.This results suggest can suppress neurovascular growth after the K5 shot, and not influence the vascular endothelial cell precursor.
Embodiment 7 K5 are to amphiblestroid safety experiment
Laboratory animal is with embodiment 3,4.
For determining toxicity and antigenicity, K57 has checked retinal structure after day in injection.Not having the infiltration of tangible Histological change or inflammatory cell takes place.In addition, blood vessel can not detectedly change after injection in the normal retina.This discovery prompting: K5 can not cause retina or normal blood vessels toxicity under used concentration.
Embodiment 8 usefulness K5 carry out the inhibitory action of animal in-vivo tumour
Laboratory animal: totally 30 of tumor bearing nude mices (inoculation human lung carcinoma cell), divide 3 groups, every group of 10 Mus.1 group: K5 tumor in-situ injection group; 2 groups: K5 lumbar injection group (positive controls); 3 groups: normal saline tumor in-situ injection (blank group).
The experiment medicine: people's K5 protein phosphatase buffer of recombinating, concentration is 3 micrograms/microlitre
Experimental technique: the in-situ injection of carrying out tumor for 1 group of mice with tumor is subjected to reagent thing 3 micrograms/microlitre/150 gram body weight; 2 groups of lumbar injections that carry out same dose; 3 groups of in-situ injection are with the normal saline of volume.Weekly injection after 4 weeks, is taken off tumor, carries out the lump size and analyzes and morphologic analysis.
Experimental result: the tumor of in-situ injection group is significantly less than abdominal cavity and normal saline injection group, and gross tumor volume is on average less than 1/3.The result shows, carries out in-situ injection to the obvious inhibitory action of tumor tool with K5.
Claims (2)
1. the application of people's recombinant plasminogen K5 fragment in the medicine of preparation treatment and new vessels generation relevant disease.
2. claim 1 is described generates relevant disease with new vessels, comprises retinopathy and pulmonary carcinoma.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101204579B (en) * | 2006-12-14 | 2011-07-27 | 复旦大学 | Application of regrouped human minute plasminogen on process for preparing the medicine curing eye disorders |
| CN108210898A (en) * | 2016-12-15 | 2018-06-29 | 深圳瑞健生命科学研究院有限公司 | Drug of tissue damage and application thereof caused by the fatty abnormal deposition of prevention and treatment |
| US11207387B2 (en) | 2016-12-15 | 2021-12-28 | Talengen International Limited | Method and drug for preventing and treating obesity |
| US11478535B2 (en) | 2016-12-15 | 2022-10-25 | Talengen International Limited | Method for preventing and treating fatty liver |
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2001
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101204579B (en) * | 2006-12-14 | 2011-07-27 | 复旦大学 | Application of regrouped human minute plasminogen on process for preparing the medicine curing eye disorders |
| CN108210898A (en) * | 2016-12-15 | 2018-06-29 | 深圳瑞健生命科学研究院有限公司 | Drug of tissue damage and application thereof caused by the fatty abnormal deposition of prevention and treatment |
| CN108210896A (en) * | 2016-12-15 | 2018-06-29 | 深圳瑞健生命科学研究院有限公司 | Prevent and treat drug of hyperlipidemia and application thereof |
| US11207387B2 (en) | 2016-12-15 | 2021-12-28 | Talengen International Limited | Method and drug for preventing and treating obesity |
| US11478535B2 (en) | 2016-12-15 | 2022-10-25 | Talengen International Limited | Method for preventing and treating fatty liver |
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