Study On The Inhibitory Effect Of Recombinant Endostatin Protein On Corneal Neovascurization

PART ONESTUDY ON THE INHIBITORY EFFECT OF RECOMBINANT ENDOSTATIN PROTEIN ON CORNEAL NEOVASCURIZATIONOBJECTIVEEndostatin is a widely-known endogenous angiogenesis inhibitor.It can strongly inhibit proliferation and migration of endothelial cells.It also causes apoptosis of endothelial cells.In recent years,the studies on endostatin mainly focus on its inhibiting the growth of tumor. There is few reports on application in other diseases associated with neovascurization.Corneal neovascurization can induce losing sight,also can induce corneal transplantation rejection,but mere are no specific drugs for treatment The aim in Part I is to study on the inhibitory effect of recombinant endostatin protein expressed by Pichia pastoris on corneal neovascurization. MATERIALS AND METHODS1. Use RT-PCR to acquire human endostatin cDNA from human embryo liver and prepare clone plasmid pGEM-En containing human endostatin gene through T-A clone.2. Construct recombinant expression plasmid containing human endostatin gene using secretory expression plasmid pGAPZ_αA and transform into Pichia pastoris GS115 through electrical shock.3. Select positive recombinant plasmid using SDS-PAGE and determined by Western Blot;Purify protein using Heparin affinity column and detect the inhibitory effect of recombinant endostatin on ECV-304.4. Prepare CNV animal models by stitching cornea and alkaline burn. On the first day after stitching cornea and alkaline burn, recombinant endostatin 0.2ml was injected into subconjunctival tissues, one time every other day,eight times in total.On the fourth day, seventh day,tenth day,thirteenth day after treatment, the lengths of new blood vessels in cornea were respectively measured with a pair ofcompasses.The growth state of new blood vessels was took pictures with digital camera,these pictures were analysed by software Immage-Pro Plus. On sixteenth day, these rabbits were killed ,the eye balls and other internal organs of the body were excised for pathological detection.In the mean time, the expressin of VEGF was detected by immunohistochemical assay, the quantity of microvessels was observed under microscope.RESULTS1. Using PCR to determine contracted clone and expression plasmid,one specific band at the site of 550bp was acquired.Two band were acquired using Not I and EcoR I disgestion.This was consistent with expected results.The result of DNA sequencing assay was totally correct.2. Linear pGAPZaA-En transformed GS115 by electrical shock, the result of 12% PAGE showed the culture supernatant of two positive recombinant clone(Bl-2,Bl-3) had one specific protein band at the site of 31-43KD.B1-3 had higher expression quantity on the fourth day.The result of Western Blot showed culture supernatant of Bl-3 had one specific protein band at the site of 31-43KD.3. The culture supernatant of Bl-3 on the fourth day was purified through Sepharose G25 and Heparin affinity column,two protein peak were acquired through washing by 0.5M NaCl,lM NaCl.The concentration of protein in 1M NaCl washing liquid was 50ng/ml.4. The results of MTT showed that concentrated supernatant of Bl-3 could effectively inhibit the growth of endothelial cell(ECV-304),the highest inhibition ratio was 74%. 1M NaCl washing liquid also could inhibit the growth of endothelial cell(ECV-304).The inhibition ratio was 86%.But they both couldn't inhibit the growth of HcpG2.2.15 cells/This affirmed mat endostatin could inhibit selectively the growth of vascular endothelial cell5. Corneal stitchingtAbout the lengths and areas of corneal neovascurization on the seventh day.the tenth day.the thirteenth day,there were significantly difference beween the group 1 of endostatin treatment (SOug/ml) and normal saline controlgroup(P< 0.05).But there weren't significantly difference beween the group 2 of endostatin treatment (25ug/ml) and normal saline control group(P>0.05).The results of pathology showed new blood vessels in the group 1 of endostatin treatment were fewer than that in control group.But there were obviously difference in other internal organs of the body beween the group 1 of endostatin treatment and control group.6. Alkaline burn: The lengths and areas of comeal neovascurization on the seventh day,the tenth day.the thirteenth day in endostatin treatment group (50ug/ml) were significantly fewer than normal saline control group(P< 0.05).The results of immunohistochemical assay showed the express quantity of VEGF in corneal matrix and the quantity of microvessels decreased significantly in endostatin treatment group(P< 0.05).CONCLUSION1. Clone plasmid and expression plasmid containing human endostatin gene are constructed successfully and expressed in Pichia pastoris Activated recombinant endostatin protein is acquired through purified by Heparin affinity column.2. Recombinant endostatin protein can effectively inhibit corneal neovascurization induced by stitching corneal or alkaline burn in vivo of animal.This will provide new strategy for prevention and treatment of CNV in clinicPART TWOSTUDY ON THE INHIBITORY EFFECT OF RECOMBINANT a-IFN COMBINED WITH LIPOFECTIN MEDIATED ENDOSTATIN GENE ON CORNEAL NEOVASCURIZATION INDUCED BY ALKALIBURN OBJECTIVEIn part I of this study,We have confirmed the inhibitory effect of endostatin on corneal neovascurization.But we also find some defects,such as short half-life, needing repeat injection.The aim of this part is to study on inhibitory effect of lipofectinmediated endostatin gene on cornea! neovascurization.We also study the inhibitory effect after combined with another antiangjogcnesis drug-rccombinant a-IFN applied in clinic. MATERIALS AND METHODS1. Construct recombinant plasmid (pVAX-sEn) expressing secretory human endostatinby pVAXl authorized by FDA in clinical trial.2. Transfcct human ovarian epithelial cell lines(3AO) mediated by lipofectin.Detect theexpression of endostatin in supematants by ELISA.Detect the inhibitory effect of pVAX-sEn on ECV-204 by MTT.3. Using pEGFP-Nl containing enhanced GFP gene as reporter plasmid, inject it intosobconjunctival tissue mediated by lipofectin,observe the expression of GFP.4. Prepare CNV animal model by alkaline burn.Observe the inhibitory effect of lipofectin mediated endostatin gene on coraeal neovascurization induced by alkaline burn. The inhibitory effect after combined with another antiangiogenesis drug-recombinant a-IFN applied in clinic is observed as well.RESULTS1. Endostatin gene with signal sequence of human IgG r chain was amplified by PCRand cloned into eukaryotic vector pVAXl .Constructed recombinant secretory expression plasmid was named as pVAX-sEN. The result of 2% gel clectrophoresis showed two specific binds at the site of 3kb and 610bp after pVAX-sEN was digested by Kpnl and EcoRI. DNA sequencing analysis indicated both the sequence of endostatin and signal peptide were in accordance with that of in the GeneBank.2. The result of ELISA showed the expression quantity of endostatin in transfectedsupernantant was 201ng/ml.The result of MTT showed pVAX-sEn transfection supernatant could effectively inhibit the growth of ECV-204,the highest inhibitory ratio was 42%.But it had no effect on 3AO.3. Using lipofectin mediated pEGFP-Nl gene to transfect corneal of rabbit by subconjunctival injection.The result of laster cofocusial microscope showed mere was expression of GFP in corneal,iris,chroid and retina of experimental group.But there wasn't expression of GFP in control group. The result of HE showed mere were significantly difference beween corneal,iris,chroid and retina tissues ofexperimental group and those of control group.This affirmed the method of which lipofectin mediated gene transfected cornea of rabbit by subconjunctival injection was practicable and secure.4. The lengths and areas of corneal neovascurization on the seventh day,the tenth day.the thirteenth day in endostatin gene treatment group (20ug) were significantly fewer than pVAXl and normal saline control group(P< 0.0S).But there wasn't significantly difference among endostatin gene treatment groupC lOug), PVAXl and normal saline control group (/*>0.05) .Combine recombinant a-IFN with endostatin gene to treat corneal neovascurization,the results showed the lengths and areas of corneal neovascurization on the seventh day,the tenth day,the thirteenth day in combined treatment group were significantly fewer than endostatin gene treatment group (20ug) and a-IFN treatment group (P< 0.05).The lengths and areas of corneal neovascurization on the seventh day.the tenth day.the thirteenth day in a-IFN treatment group were fewer than pVAXl and normal saline control group(P< 0.05).But there wasn't significantly difference between endostatin gene treatment group (20ug) and a-IFN treatment groupCONCLUSION1. Construct successfully recombinant secretory expressing plasmid pVAX-sEn containing human endostatin gene.2. Lipofectin mediated pVAX-sEn gene can effectively inhibit the growth of ECV-204 in vitro.3. Recombinant a-IFN combined with lipofectin mediated PVAX-sEn gene can effectively inhibit corneal neovascurization induced by alkaline burn.This will provide experimental foundation for treatment of CNV in clinic.4. The method of which lipofectin mediated gene transfected cornea of rabbit bysubconjunctival injection is practicable and secure. PART THREESTUDY ON INHIBITORY EFFECT OF RECOMBINANT a-IFN COMBINED WITH LBPOFECTIN MEDIATED ENDOSTATIN GENE ON THE GROWTH OF OVRIAN CANCEROBJECTIVEInhibition of angiogenesis is the key to the question for the treatment of primary tumor and controlling of metastases. The aim of mis part is to study the inhibitory effect of recombinant a-IFN combined with lipofectin mediated endostatin gene on the growth of tumor in nude mice bearing with 3AO.MATERIALS AND METHODS1. Using 3AO to inoculated subcutaneously to the axilla of nude mice to prepare ovarian cancer animal model,using recombinant a-IFN combined with lipofectinmediated pVAX-sEN to inject intratumorly, two times one week^bur times totally,the inhibitory effect of the growth of ovarian cancer was observed.2. Using tumor tissue and important internal organs of the body for pathological detection,the effect of pVAX-sEn transfection on tumor tissue and internal organ of the body was observed.RESULTS1. Using 3AO to inoculated subcutaneously to the axilla of nude mice,7-10 day later, the tumor became to grow.the ratio of tumor growth was 100%.2. The tumor volumes in the group of using recombinant a-IFN combined with lipofectin mediated pVAX-sEn were significantly less than mat in pVAX-sEN treatment group;The tumor volumes in pVAX-sEN treatment group were significantly less man that in normal saline control group and pVAXl treatment group (PO.05). But beween pVAXl treatment group and normal saline control group,there was no significantly difference (P>0.05) .3. HE stain in tumor tissue showed mat mere were obviously necrosis tumor cells,fewer blood vessels in the group of using recombinant a-IFN combined with PVAX-sEn,but there were flourishly growth tumor cells and plenty of blood...