The Experimental Study Of Preventing Taget Organs Thrombosis By Locally Transfected Tissue-Type Plasminogen Activator Gene

Part I The Experimental Studies of Preventing VeinGrafts Restenosis by Locally Transfected Tissue-TypePlasminogen Activator GeneExperiment I Construction and Indentification of pcDNA3.1-Myc-His B(-)/tPA Gene Recombinant Eukaryotic Expressing VectorObjective To construct a recombinant eukaryotic expressing vector pcDNA3.1-Myc-His B(-)/tPA including functional region of human tissue-type plasminogen activator(tPA) gene to provide a basis for further study on the therapy of thrombotic disease.Methods The tPA gene fragment was obtained from PBS/tPA cloning vector by restricting enzyme digesting and cloning into pcDNA3.1-Myc-His B(-) eukaryotic expression vector.Analysis by restricting enzyme digesting and DNA sequencing was carried out to demonstrate the sequence of the plasmid. Results Restrictive enzyme(KpnI/NotI) digestion analysis showed that the 2. 2kb and5. 5kb bands were coincidence with tPAcDNA and vector DNA respectively.Sequencing result revealed the sequence of tPA genome was identical with that in GenBank. Conclusions The recombinant eukaryotic expression vector pcDNA3.1-Myc-HisB(-)/t-PA is successfully constructed.Experiment II The Change of Plasmin Activity in Human Umbilical Vein Endothelial Cells with Transfected tPA GeneObjective To observe the change of plasmin activity in human umbilical vein endothelial cells(HUVEC) with transfected tissue-type plasminogen activator (tPA) gene.Methods Recombinant plasmid pcDNA3.1-Myc-His B(-)/tPA was transfected into HUVEC line cells mediated by lipofectamine2000. The positive clones were obtained by the screen of G418. The transcription and expression of tPA gene were investigated by RT-PCR and Western-Bloting respectively. The levels of human tPA antigen in culture media was measured suing enzymelinked immunosorbent assay.The tPA activity was measured by chromogenic substrate assay Results :A positive clone cells from which transcripted the mRNA of t-PA gene was obtained by RT-PCR. In the transfected tPA gene group,vector group and blank group,the levels of tPAmRNA was 0.94± 0.20; 0.32± 0.15 and 0.36± 0.16 respectively, the contain of tPAmRNA in the transfected t-PA gene group was significantly higher than that in the control groups. The secretion of tPA antigen was 612.36±116.12ng/10⁶cells/24h, 59.17 ± 21.18 ng/10⁶cells/24h and 57.42 ±18.83 ng/10⁶cells/24h respectively, the levls of human tPAantigen in transfected tPA gene group was significantly higher than that in control groups. The tPA activity in the media was 57.95 ±11.22IU/10⁶cells/24h, 4.65 ± 1.40IU/10⁶cells/24h and 4.68±1.31/10⁶cells/24h respectively , the tPA activity was increase significantly in the transfected tPA gene group than that in the control groups.The tPA protein could be detected with anti-myc antibodies in transfected tPA gene group and could not be detected in the control group. Conclusions: The recombinant eukaryotic expression vector pcDNA3.1-Myc-His B(-)/tPA can stable express tPAcDNA in transfected hUVEC cell strains and can secret compt tPA protein. Experiment III The Effects of Transfected tPA Gene on Vascular Smooth Muscle Cells Proliferation and Migration in VitroObjective To investigate the effects of transfected tPA gene on vascular smooth muscle cell proliferation and migration in vitro and to provide a basis for further study on preventing vein graft restenosis.Method Recombinant plasmid pcDNA3.1-Myc-His B(-)/tPA was transfected into mice vascular smooth muscle cells mediated by Iipofectamine2000. The transcription and expression of tPA gene were investigated by RT-PCR and Western-Bloting respectively. The levls of human tPA antigen in culture media was measured suing enzymelinked immunosorbent assy. The tPA activity was measured by chromogenic substrate easy. The cell attachment assay,cell proliferation assay and cell migration assay were carried out to detect the cell attachment, proliferation and migration respectively. Result RT-PCR and Western-Blot confirmed the expression of tPAmRNA and the presence of tPA protein in the transfected tPA gene group,and not in the control groups.The tPA activity detected in the transfected tPA gene group was 26.6 ± 7.02 IU/10⁶cells/24h, and could not detect in the control groups.The tPA contains in the transfected tPA gene group,vector group and blank group were 518.38±125.32 ng/10⁶cells/24h, 12.53±4.27 ng/10⁶cells/24h and 13.21 ± 5.02 ng/10⁶cells/24h respectively,the tPA contain was significantly higher than that in the control group.There were no difference observed in cell attachment,proliferation and migration among three groups.Conclusion The expression of high levels of tissue plasminogen activator does not specifically affect VSMC attachment,proliferation and migration. Experiment IV The Effects of Tissue-type Plasminogen Activator Gene Therapy on Restenosis of Vein GraftsObjective To observe the effects of in vivo local expression of recombine human tissue-type plasminogen activator (tPA) gene on the thrombosis and neointima formation in vein grafts and identify the mechanism. Methods Jugular vein-to-artery bypass grafting was performed on 72 New Zealand white rabbits. Rabbits was devided into 3 groups according to the different processing methods, including transfected tPA gene group, transfected empty vector group and saline group .Samples of vein graft was harvested at different time points after sugery.The expression of tPA gene in vein graft was detected by RT-PCR and Western-Blot essay. The tPA activity was measured by chromogenic substrate essay. The Cr51 labeled platelets accumulation in vein grafts was detected by platelet counting.The IH was compared after operation.Results At the 2^nd, 5^th, 14^th,28^th day after operation,RT-PCR and Western-Blot confirmed the expression of tPAmRNA and the presence of tPA protein at the site of gene transfer.The tPA activity detected on the 2^nd,5^th, 14^th,28^th day in experimental group was 370.63±59.44 ,344.13±48.47 , 252.87±51.80 and 161.75±68.94 U/g respectively, but no tPA activity was detected on the 60^th day and in the control groups.The number of platelets accumulation in the vein grafts in the experimental group,empty vector group and saline group was 85.04±21.58×10⁶ ,225.87±85.13×10⁶ and 211.57±78.02×10⁶ respectively/The number of platelets accumulation in the experimental group was significantly fewer than that in the control groups.Morphometric analysis showed that in the tPA gene group,intimal hyperplasia was markedly reduced than those in the control groups.At the 2^nd,5^th ,14^th,28^th,60^th day, the ratio of PDGF positive cells in the vein grafts with transfected gene group was lower than that in control groups. At the 2^nd ,5^th ,14^th,28^th day, the ratio of TGF-β1 positive cells in the vein grafts with transfected gene group was lower than that in control groups,at 60^th day,the difference was not sinnificant.Conclusion Local expression of tPA gene in vein graft significantly inhibited the accumulation of platelets and thrombosis., reduced intimalhyperplasia,therefore ,prevented restenosis afrer bypass graft.Part II The Effects of Gelatin Coating Dacron Patch Containing tPAcDNA on Plasminogen Activaty in Rabbit LeftAtrium BloodObjective To evaluate the effects of locally applied tPA gene on plasma plasmingen activity in left atrium blood. Methods 36 New Zealand white rabbits was devided into 3 groups according to the different processing methods, including transfected tPA gene group(implant gelatin coating Dacron patch containing tPAcDNA in left atrium), transfected empty vector group (implant gelatin coating Dacron patch containing empty vector) and blank group (implant gelatin coating Dacron patch without gene ), on the 3^th, 14^th day, the rabbits atrium muscle containing the implanted Dacron patch was take out after operation, The expression of tPA gene in left atrium muscle was detected by RT-PCR and Western-Blot essay. The tPA activity of plasma plasmingen in left atrium and peripheral blood was measured by chromogenic substrate essay.Results At the 3^th, 14^th day after operation, RT-PCR and Western-Blot confirmed the expression of tPAmRNA and the presence of tPA protein at the site of gene transfer.The tPA activity in left atrium blood detected on the 3th, 14^th day in experimental group was significantly higher that in control groups.Conclusion Gelatin coating Dacron patch is feasible to be the carrier of gene transfection. Locally applied tPA gene in atrium muscle can increase tPA activity in left aterium blood.