| Objective: To study the mechanism of proliferation and apoptosis of VSMC, VEC and vein graft neointimal hyperplasia, and then explore the new method of effective prevention and control vein graft stenosis and to provide a theoretical basis for the prevention of thrombosis. Construct targeting rat Pik3cb eukaryotic expression vector by SM22αand KDR specific promoter combined with RNA interference principle and transfected it into vein grafts through delivery system of the Pluronic F-127.Methods: The sense and antisense RNA oligonucleotide strands targeting Pik3cb mRNA were designed and synthesized individually according to PI3K p110βsubunit gene Pik3cb mRNA sequence (NM: 053481) in rat Genbank database, then cloned it into pGenesil-1 plasmid expression vector with SM22αp/e, KDR p/e and CMV promoter, and named them as SM22α-Pik3cb-shRNA, KDR-Pik3cb-shRNA and CMV-Pik3cb-shRNA, respectively. Modified rat vein-to-artery interposition models using the autologous branch of jugular vein were constructed. Jugular vein grafts were treated with 25% Pluronic F-127 only (group A, n = 30), plasmid encoding shRNA targeting Pik3cb p110βsubunit (KDR-Pik3cb-shRNA, SM22α-Pik3cb-shRNA and CMV-Pik3cb-shRNA, groups B, C and D, n = 30, respectively), KDR-Pik3cb-shRNA+SM22α-Pik3cb-shRNA (group E, n = 30), or wortmannin (Group F, n = 30). Specimens were harvested at 1, 3, 7, 14 and 28 days post surgery to assess jugular vein graft neointimal hyperplasia.Immunohistochemical staining was performed with primary antibodies phosphor-mTOR (Ser2448), as well as TUNEL method to evaluate the antiproliferative effects of shRNA. Otherwise, a separate group (n = 18, 3 for each group) received the same treatment as above were killed on postoperative day 3 for assays to evaluate relative amount expression of Pik3cb mRNA.Results: Rat vein-to-artery interposition models using the autologous branch of jugular vein were uccessfully constructed. Observing patency rate of vein graft, the positive control group compared with groupB were statistically significant (x2 = 4.593, P < 0.05), and group B was the lowest (23.3%),the positive control group compared with groupC were statistically significant(x2 = 9.32, P < 0.01), and group C was the highest patency rate (86.7%).Three days after surgery, detected Pik3cb mRNA relative expression by real time polymerase chain reaction, the positive control group and the plasmid transfected groups decreased significantly than that in the control group (F = 386.3, P < 0.01). Group B, C, D, E and F were decreased by 46.8%, 73.3%, 81.2%, 64.2% and 68.4%, respectively.One and three days after surgery, neointimal thickness in every group was no significant difference; 7 days after surgery, neointimal thickness in positive control group and the plasmid transfection groups compared with the control group there were significantly increased (F = 82.59, P < 0.01),14 days after surgery, neointimal thickness in positive control group and the plasmid transfection groups compared with the control group there were significantly increased (F = 325.09, P < 0.01), 28 days after surgery, neointimal thickness in positive control group and the plasmid transfection groups compared with the control group there were significantly increased (F = 483.76, P < 0.01). Twenty-eight days after surgery, the neointimal thicken of 10.4 times as compared to day one post-surgery in the control group; groups B, C, D, E and F were 11.6 times, 6.0 times, 7.1 times, 6.8 times and 6.5 times, respectively.Vein graft phosphorylation mTOR (Ser2448) positive expression area in positive control group and in the plasmid transfection groups were significantly lower than that in the normal control group, difference was statistically significant (F=50.41,P < 0.05); at 28 days, the percentage of positive area of group B, C, D, E and F were 0.93%, 0.69%, 0.49%, 0.47% and 0.43%, respectively, while the positive control group was 1.48%.At different time points, apoptotic cells of vein graft tissue were significantly higher in the positive control group and the plasmid transfection groups than that of the control group, difference were statistically significant (F=134.38,P < 0.05); at 14 days, positive apoptotic cells in all groups reached the peak, the percentage of positive area of the control group was 1.27%; group B, C, D, E and F were 1.95%, 2.43%, 3.18%, 3.06%, 2.94%, respectively.Conclusion: Rat carotid vein-carotid artery interposition model is a ideal model for study vein graft stenosis in coronary artery bypass graft; SM22α-Pik3cb-shRNA can attenuate intimal hyperplasia, the mechanism may be downregulation of PI3K-Akt-mTOR signaling pathway to inhibit vascular smooth muscle cell proliferation and promote it's apoptosis; KDR-Pik3cb-shRNA can promote thrombosis of the vein graft and increased intimal thickness, and its mechanism may be downregulation of PI3K-Akt-mTOR signaling pathway to inhibit endothelial cell proliferation and then delay endothelialization of the intima.
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