Study On The Prevention Of Venial Bypass Restenosis By Transfection Of Endothelial Nitric Oxide Synthesase Gene

Although the self great saphenous vein is the most widely used material in coronary artery bypass surgery(CABG),the lower unobstruction rate always harass the surgeons. The mechanism of post-CABG restenosis remains unclear. Now, it is generally accepted that multiple factors such as surgery injury, ischemia duration of graft vein, and arteriation are involved. These factors may induce damage, isolation of endothelial cells, and aggregation of platelet and white cells which promote formation of thrombus and hyperplasia of inner membrane. The traditional drugs have no effect on prevention of veinous thrombus formation and restenosis. Restenosis rate after vein bypass is higher compared with that after artery bypass. Previous studies indicated post-CABG vein restenosis was closely correlated with abnormal nitride oxide (NO) function. Endothelial NO synthesase (eNOS) plays the important role in the synthesis of endothelial NO. In the present study, we investigated localization of eNOS in three kinds of grafts by using immunohistochemistry method and found content of eNOS was higher in radial artery and inner mammary artery than that in great saphenous vein. So, we speculate that the higher expression of eNOS in inner membrane and medial muscle may be the indicator of favorable unobstruction. eNOS gene recombination by transgene technique is expected to reconstruct eNOS function,and hence promoting local release of eNOS to prevent post-CABG restenosis. In the current study, eNOS gene was transfected into isolated great saphenous vein and animal vein bypass using replication deficient adenovirus vector. By using FISH and immunohistochemical methods, we studied the transfection efficiency, expression of eNOS, and impact of eNOS gene transfection on hyperplasia of inner membrane of vessels and evaluated the feasibility and validity of eNOS gene transfection to provide basis for gene therapy in clinical practice. The paper included the following four parts.Part I:Amplification and purification of AdCMVeNOSObjective: To identify, amplify, and purify AdCMVeNOS and AdCMV. Mehtods: AdCMVeNOS and AdCMV were used to transfect HEK293 cell to amplify the two adenovirus vectors. Vectors were purified through CsCl density gradient centrifugation after cell showed sign of pathological alterations. Adenovirus titration was determined by counting the number of plaques. DNA was extracted from AdCMVeNOS-infected HEK293 cells and polymerase chain reaction (PCR)used to detect the eNOS gene fragement .Results: PCR results showed the 1770bp eNOS gene fragment was integrated into AdCMV. Titration of AdCMVeNOS and AdCMV was more than 4.8 xl0~10pfu/ml after purification.Conclusion: AdCMVeNOS carried the eNOS gene fragment. We obtained high-titration recombinant adenovirus.Part II: Immunohistochemical localization of eNOS in three kinds of vessels grafts used in CABGObjective: To investigate the expression and localization of eNOS in three kinds of vessels used in CABG, radial artery(RA), inner mammary artery(IMA) and great saphenous vein(SVG) using immunohistochemistry technique. Methods: The discarded left IMA(LIMA), RA, and SVG specimens for 20 patients were obtained. LM was used to observe the staining of eNOS and results were analyzed using image analysis software. Results: The marked expression of eNOS was observed in inner membrane of three kinds of vessels, in media layer of. eNOS expression was observed in LIMA and RA. The grey value for LIMA was higher than that for RA and SVG(P<0.05), in inner membrane, while in media layer, the value for LIMA was higher than that for RA(P<0.05).Conclusion: The eNOS was expressed in inner membrane of LIMA,RA and SVG, in media layer in LIMA and RA. Expression difference among the materials may be the factor affecting the long term therapeutic effect.Part III: Study on the transfection of endothelial NO synthesase gene into cultured human great saphenous veinObjective: To construct cultured human great saphenous vein model in vitro, then introduce AdCMVeNOS and detect the expression of eNOS and investigate mechanism of hyperplasia of inner membrane. Methods: The residual great saphenous veins from 20 patients undergone CABG were obtained and cut into 5mm blood circle, which was divided into group A,B and C, each group having 6 samples. Group A was control, group B was empty adenovirus vector transfected, group c was study group. Samples of group B and C were incubated for lh in empty adenovirus andAdCMVeNOS solution, and then taken out and cultured for 14. Additionaly, 18 blood circles were cultured for 3, 7 and 14 days to observe the growth of inner membrane of vessels. Oligonucletide probe and high-sensitivity marking technique was used to detect the expression of exogeneous expression of eNOS. Image analysis software was used to evaluate the hyperplasia of inner membrane and media layer. SABC method was used to detect the expression of eNOS, PCNA, alpha-actin. TUNEL to detect the apoptosis. Proliferation and apoptosis rate was evaluated. Results: We observed hyperplasia of inner membrane of great saphenous vein during culture duration. Inner membrane and media layer showed increase in depth.alpha-actin positive smooth muscle cells, PCNA positive proliferation cells and TUNEL positive apoptosis cells were observed in newborn inner membrane 14 days after culture. ENOS mRNA and protein expression was observed in group C. Group B showed no change in depth of inner membrane and media layer as compared with that in control group. Inner membrane and media layer was decreased by 33.6% and 11.6%, respectively in group C. The ratio of inner membrane to media layer was decreased by 53.8% compared with that in control group. Group C showed significant reduction in proliferation ability and enhancement in apoptosis. Conlcusion: Migration of smooth muscle cells, proliferation, and apoptosis during hyperplasia of inner membrane may all participate in the reconstruction of inner membrane. We successfully transfected AdCMVeNOS into inner membrane and media layer of cultured human great saphenous vein. ENOS was significantly expressed in target tissue, which may inhibit hyperplasia of inner membrane.Part IV: Establishment of animal model of vein bypass graft and study on transfection of vein bypass with AdCMVeNOSObjective: To construct rabbit model of vein bypass graft, observe pathological changes in vein bypass and investigate the mechanism of reconstruction of vein bypass. To transfect bypass graft vein with AdCMVeNOS and detect eNOS expression in jugular vein to explore the approaches for prevention of restenosis of grafted vessels.Mehtods: Total 30 rabbits were used. The unilateral general jugular vein and outer jugular vein were isolated and anastomosed side by side. The part outside anastomose and middle part of general jugular vein anastomose were ligated to make arterial blood pass through outer jugular vein. Vessel bypass was obtained 3days,l,2,4,and 8 weeks after surgery. Bypass graft model of general jugular vein and outer jugular vein of rabbit was used. 18 rabbits were randomly divided into 3 groups. Group A was control. Group B was...