Experimental Study On The Effect Of Local Transfection Of C - Type Natriuretic Peptide On Restenosis After Angioplasty

PART1Reconstruction and sequencing of rabbit C-type natriureticpeptide gene eukaryotic expression vectorObject For angioplasty investigation of gene therapy,to reconstruct rabbit C-type natriuretic peptide gene recombinant eukaryotic expressive vector.Methods By RT-PCR from rabbit abdominal aorta and sub-cloned into pMD 18-T vector,the full coding domain sequence of rabbit CNP gene fragemeng was cloned.After using enzyme Pst â…  digestion negative pMD 18-T/CNP,the targeted DNA fragment digested with restriction enzyme Hind III and Kpn â… ,was directional cloned into eukaryotic expressive plasmid pEGFP-Nl.Then,the recombinant eukaryotic expression plasmid pEGFP-Nl/rCNP was identified with double enzyme digestion and sequence determination.Results It was donfirmed that the rabbit CNP gene was accurately cloned and inserted into the accurate sites between Hind â…¢ and Kpn I in pEGFP-N1 without mutation and transposition.Conclusion The successive reconstruction of eukaryotic expressive plasmid containing genes of rabbit C-type natriuretic peptide established the research foundation for CNP gene therapy.PART 2Construction of rabbit CNP gene eukaryotic expression vector and its expression in human umbilical vein endothelial cellsObjective To dectect the rabbit CNP gene eukaryotic expression vector expression in human umbilical vein endothelial cells in vitro.Methods Rabbit CNP gene was inserted into an eukaryotic expression vector pEGFP-N1 carrying the enhanced green fluorrescent protein,to generate a new plasmid pEGFP-N1/rCNP. The identified recombinate pEGFP-N1/rCNP eukaryotic expression vector was transfected into human umbilical vein endothelial cells with Lipofectamin TM 2000 liposome.Results The eukaryotic expression vector containing rabbit CNP gene was constructed sucessfully.EGFP report gene and CNP gene were expressed in human umbilical vein endothelial cells transfected with the recombinate plasmid pEGFP-N1/rCNP.Conclusion The recombinate vector pEGFP-N1/rCNP is constructed and rabbit CNP gene can be normally expressed in human umbilical vein endothelial cells.PART 3Local transfect of C-type natriuretic peptide gene prevents vascular restenosis in rabbit model of vascular injuryObjective To detect the expression of rCNP recombianant plasmid expression in rabbit Iliac artery and observe its effect on the prevention of restenosis after angioplasty.Methods Recombinant eukaryotic expression plasmid pEGFP-N1/rCNP was transfected with intraluminal compressive method in Iliac artery of rabbit anastomosis restenosis model.The transfect efficiency was determined with blank control pEGFP-N1.We using the RT-PCR,Western blot and immunohistochemistry method to observe the expression of pEGFP-N1/rCNP in the rabbit illac arteries.Respectively, re-endothelialization,intimal hyperplasia were detected with pathological methods and immunohistochemistry.Results It was confirmed that the recombinant eukaryotic expression plasmid pEGFP-N1/rCNP can effectively express in Iliac artery of rabbits.CNP mRNA and protein expression were significantly increased. Expression of CNP and blank pEGFP-N1 group was obviously reduced.Moreover,it was observed that the repaired in transfected group was enhanced;hyperplasia significantly and restenosis rate was reduced.Obviously decreased,the rate of restenosis after angioplasty.Conclusion Restructuring of CNP eukaryotic expression plasmid PEGFP-N1/ rCNP can be efficiently expressed and introduced in anastomosis restenosis model of rabbits illac artery.CNP peptide was capable of inhibiting restenosis with promoted re-endothelialization and inhibited hyperplasia.