Dissertation > Excellent graduate degree dissertation topics show

Gene Therapy with Endothelial Nitric Oxide Synthase Protects Aortic Allografts from Arteriosclerosis

Author: CaoHua
Tutor: LiaoChongXian；ChenDaoZhong
School: Fujian Medical
Course: Surgery
Keywords: Gene therapy Endothelial nitric oxide Arteriosclerosis Transplant
CLC: R654.2
Type: PhD thesis
Year: 2003
Downloads: 36
Quote: 0
Read: Download Dissertation

Abstract

Allograft vascular sclerosis disease is a major cause of death in heart transplant patients with long-term survival. Complex pathophysiological mechanisms has not been elucidated, there is no effective treatment, with emphasis on prevention. In this study, the Construction and identification of recombinant plasmid PLXSN-eNOS, liposome-mediated transfection method directly transfected into the transplant artery to explore the role of eNOS gene allograft prevent hardening of the arteries. The study is divided into two parts: the first part of the application of PCR and restriction enzyme digestion technology constructed recombinant plasmid PLXSN-eNOS were identified; second part of the application by RT-PCR confirmed plasmid PLXSN-eNOS whether transplanted successfully transfected vascular, and by The detection of the indicators to explore the role of eNOS gene therapy for prevention of hardening of the arteries of the allograft. First inner cutting enzyme ECOR Ⅰ and hind Ⅲ respectively plasmid PGEM-3zf-of eNOS and Pblue dual enzyme, carrier Pblue and eNOS purpose gene carried ligation reaction constructed plasmid PLXSN-of eNOS experiment constructed plasmid Pblue-of eNOS; Second inner cutting enzyme ECOR Ⅰ and Xho Ⅰ respectively after plasmid pBlue-of eNOS and PLXSN dual digestion, vector PLXSN and eNOS purpose gene carried ligation reaction, construct plasmid PLXSN-of eNOS; final plasmid PLXSN-of eNOS double identification: PCR identification electrophoresis formed 760KB a band; endonuclease ECOR Ⅰ and Xho Ⅰ double digestion electrophoresis to form two bands. PCR and restriction analysis proved the plasmid PLXSN-eNOS build success. Liposomal transfection PLXSN-eNOS-LF2000 transplant carotid artery experiments, the the SD rats carotid artery orthotopic transplantation in Wistar rats Application 11-0 without injury Line, rats transplanted with the microsurgical technique carotid artery anastomosis. The experiment was divided into control and experimental groups. The control group and the experimental group for the body of the carotid artery were soaked in an eNOS PLXSN-LF2000 liposomes and pLxSN a LFZ000 liposome solution after 30min. The experiment research; (1) RT-PCR method to detect postoperative 7, 14 and 28-day experimental group transplanted artery to observe whether the eNOS gene transfection of vascular graft. (2) immunohistochemical methods to detect individual when f the Division points transplantation artery eNOS, iNOS, NF a kB expression of ICAM-1 and VCAM-l expression. (3) light microscopy and electron microscopy analysis of the the transplant vascular pathological changes, and the detection of intimal medial smooth muscle hyperplasia proportion. The experimental results show that: (1) after 14 and 28 days, the experimental group transplanted vascular RT-PCR detection, agarose gel electrophoresis image analysis can be seen two with proved exogenous gene. NOS successfully transfected with blood vessels. (2) control group eNOS and iNOS expression changes: postoperative 7, 14 and 28 days, the transplant vascular endothelial cells eNOS significantly weakened; transplant intimal iNOS expression was significantly increased, iNOS smooth muscle cells, monocytes / macrophages The positive expression of macrophages and endothelial cells, vascular graft medial smooth muscle cell iNOS expression enhanced after 1 and 7 days, but after 14 and 28 days later, and gradually reduce. The experimental group eN0s and iNOS expression changes: the transplant vascular endothelial cells eNOS expression was significantly enhanced intimal smooth muscle cells when visible expression of: intimal iNOS expression was significantly weakened; the transplant vascular media iNOS expression after 1 and 7 days weakened, but surgery after 14 and 28 days, with no significant difference compared to the control group. (3) postoperative 7, 14 and 28 days in the control group of transplant intimal expression of ICAM 1 and VCAM an obvious enhancement, the experimental group, ICAM-1 and VCAM-1 expression was decreased. (4) postoperative 7, 14 and 28 days in the control group of transplant intimal the NF kB express enhanced experimental group NF kB expression weakened. (5) light microscopy and electron microscopy transplant vascular pathological changes: the control group at 28 days after transplantation, the intimal indemnity chronic thickening, involving the whole ring intimal lumen narrowing subintimal smooth muscle cells increased significantly, large amounts of extracellular matrix formation. Middle thinning, part of the smooth muscle cells degenerate. The elastic fiber layer remain intact. The increase in the proportion of the intima-media. eNOS gene therapy, intimal hyperplasia and luminal stenosis significantly reduced, there is no significant monocyte / macrophage infiltration. Medial smooth muscle cells remain intact. Electron microscopy showed graft intimal thickening, monocyte / macrophage infiltration, the intima lower 3 contains a large number of smooth muscle cells, abundant extracellular matrix and collagen fibers, medial smooth muscle cell degeneration. Based on the above experimental results, we can draw the the following results 1.PCR and endo the enzyme ECORI and Xhol double digestion prove PLXSN eNOS plasmid was constructed successfully. ENOS 2.PLXSN a one LF20OO liposomes directly soak transfected artery transplantation. Turn 3.eNOS gene therapy to maintain normal allograft intimal eNOS expression, inhibition of iNOS expression of ICAM 1, VCAM-1 and NF-kB expression, reducing intimal hyperplasia, prevention of allograft vascular disease

Related Dissertations

CLC: > Medicine, health > Surgery > Of surgery > Cardiovascular and lymphatic system surgery > Heart
© 2012 www.DissertationTopic.Net Mobile