Adenovirus-mediated VEGF165 Gene Transfer For Preventing Vein Graft Restenosis

Objective: To detective the protein'influence expressed by VEGF165 on autologous vein graft intimal by the way of transfection of vein grafts through incubation before the operation of bypass grafts.Method: The Ad5-VEGF165-CMV-EGFP of purchase was amplified to the number of need for the virus in 293 cells, then purified it through cesium chloride (CSCL) gradient centrifugation. Identified adenovirus containing the gene by PCR and kept it frozen. Performed left femoral artery bypass surgery on rabbit using femoral vein in homemade vascular support implement auxiliary. The vein which was from the experimental group was immersed in a solution of VEGF165 adenovirus(virus titer was 1.0×109pfu/ml) and control group was immersed in the salt water. Both were required to 37℃incubation 30 minutes and flushed by warm salt water. After 2d, 4d, 6d, 8d, 10d, 12d, 14d, 3w, 4w, 5w, 6w, 7w from the experimental group and a control group, animals were sacrificed on the line grafts VEGF165 gene RT-PCR detection. The femoral vein of the endometrial thickness from the experimental group and control group was measured and statistically analyzed. Calculated intimal hyperplasia index, drawn femoral artery bypass grafting time - curve of vein graft intimal hyperplasia. Directly observed the histological changes of intima hyperplasia on the vein graft by way of HE and elastic fiber staining.Results: Ad5-VEGF165-CMV-EGFP can be amplified well in 293 cells and transfected to receive drugs for 11 days. The purification of adenovirus particles and titer can be 2.5×1011 VP / ml and 1×1010 PFU / ml after three generations of amplification. Vascular support with homemade devices could bring a lot of convenience for femoral artery bypass surgery, for example improving the success rate of anastomosis and shortening the operation time about 10 minutes. Vein graft mRNA expression was obtained after two days, which would continue for 6 weeks. Transgenic group was smaller than the control group in graft vein intimal hyperplasia through HE and elastic fiber staining, which proliferated too. Both of the results obtained was differently statistically significant by Wilcoxon signed rank test. Intimal hyperplasia index also showed the transgenic group serious than in the control group proliferation.Conclusion: Adenovirus-mediated VEGF165 gene which was incubated in vitro could inhibit intimal hyperplasia through transfecting vein grafts, which play a role in preventing restenosis.