Prevention Of Grafted Vascular Stenosis In Rats Through The Modification Of MTOR Signaling Pathway Under The Driving Of Specific SM22α And KDR Promoter/enhancer

Objectiv:To study the mechanism of proliferation and apoptosis of valscuar soomth muscle cells(VSMC),valscuar endothelial cells(VEC) and vein graft neointimal hyperplasia and explore the new method of prevention of vein neointimal hyperplasia and provide a theoretical basis for the prevention of vein graft stenosis, we transfect the specific of vascular smooth muscle cell SM22a promoter/SMHC enhancer and KDR promoter/enhancer,which are able to downragulate rat mTOR signaling pathway, into the vascular model of jugular vein grafted to carotid artery through delivery system of the Pluronic F-127.Methods:To construct the eukaryon expression vectors targeting on mTor of rats, named as SM22a-p/e-mTOR-shRNA, KDR-p/e-mTOR-shRNA, CMV-mTOR-shRNA,respectively. And to construct rat jugular vein-to-artery interposition models. Then to transfect shRNA around the jugular vein-to-artery through the plsmid delivery system of the Pluronic F-127. And we randomize rats into7groups:Group A:the group of25%Pluronic F-127(matched group, n=20); Group B:the group of SM22a-p/e-mTOR-shRNA (group of SM22a, n=20); Group C:the group of KDR-p/e-mTOR-shRNA (group of KDR, n=20); Group D:the group of SM22a-p/e-mTOR-shRNA and KDR-p/e-mTOR-shRNA half for each (group of combination, n=20); Group E:the group of CMV-mTOR-shRNA(group of CMV, n=20); Group F:the negative control empty plasmid group(n=20); Group G: wortmannin positive control group(n=20). Jugular vein grafts were with25%Pluronic F-127only(200μl), plasmid encoding shRNA(50μg in25%Pluronic F-127), wortmannin(50μg). Specimens were harvested at1,3,7,14days post surgery to assessed jugluar vein graft neointimal hyperplasia through H-E, Measured transplanted vein anastomosis proximal endometrium thickness on averge by computer image analysis system. Immunohistochemical method was performed with antibodies a-smooth actin, CD-34for observation of smooth muscle cells and endothelial cell integrity, respectively, as well as TUNEL method to evaluate the antiproliferative effects of shRNA.Results:Rat vein-to-artery models were successfully constructed using the autologous branch of jugular vein.14days post surgery, endothelial cells of matched group, group of SM22a, negative control empty plasmid were integrally. But group of KDR, group of combination, group of CMV, positive control group were not.There has difference the group of neointimal thickness(P<0.05), and the proliferation of new vascular endothelium cells was significantly higher at14days after operation in matched group, group of KDR, negative control group, and there has difference compared the group of SM22a, group of combination, group of CMV with matched group and negative control group(P<0.05).14days post operation, neointimal thickness of matched group, group of SM22a, group of KDR, group of combination, group of CMV, negative control group were increased by12.6,9.7,10.8,1.4,5.1,7.6and2.5fold than1days.14days post operation, the α-SM-actin masculine cells in matched group, group of KDR, negative control group were significantly more than group of SM22a, group of combination, group of CMV and positive control group(P<0.05). The percentage of masculine cells in group of SM22a, group of combination, group of CMV and positive control group at14days(1.48±0.09,1.05±0.54,0.97±0.14,0.95±0.37, respectively) were significantly reduced, compared with matched group, group of KDR, negative control group(5.42±0.16,3.2±0.76,3.97±0.69, respectively).As time went by, the aptosis cells of graft vein were increased in postoperation each group, it’s the highest in14days. The percentage of aptosis cells in matched group, group of KDR, negative control group(0.43%,0.2%,0.73%) were lower than group of SM22a, group of combination, group of CMV and positive control group(0.84%,2.4%,1.87%,2.78%).Conclusions: The model of Rats jugular vein branch-carotid artery is a idea for study grfat vein stenosis after coronary artery bypass grafting. SM22a-p/e-mTOR-shRNA could effectively reduce graft vein thrombosis and neointimal hyperplasia, for its just specific inhibition of vascular smooth muscle cell proliferation and promote its apoptosis, dose not affect the regeneration of the vascular endothelial cell. The KDR-p/e-mTOR-shRNA has specific effects on vascular endothelial cell to affect its repair, and then demonstrate that graft vascular endothelial regeneration to repair as soon as possible to avoid or reduce thrombosis, and thus reduce neointimal hyperplasia and stenosis degree.