Effects Of Survivin Gene Over Expression On Vascular Endothelial Cells

Periphery artery disease, characterized by peripheral artery stenosis, occlusion, embolism, is most common in lower limb arteriosclerosis obliterans. At present, the lesion of arterial wall can not be reversed by operation or drugs. The key of periphery artery disease's treatment is vascular endothelial cells, which could increase the peripheral circulation, improve the microcirculation of the blood perfusion and solve the problem of arterial long-term patency rate after periphery revascularization by proliferation, migration and formation of the bureaucratic structure or new blood vessels net. However, the activity, growth rate and update speed of mature VECs is relatively low, the ability of degrading the extracellular matrix and migration is not enough to establish collateral circulation. In conclusion, an ideal VECs equipped with a powerful ability of anti-apoptosis, proliferation and degrading the extracellular matrix, migration at the same time is most important in PAD's treatment.Survivin, a smallest molecular weight member of inhibitor of apoptosis protein family, plays an important role in regulation of anti-apoptosis and proliferation in tumour cell. SW is known as the strongest apoptosis inhibiting factor at present, which could inhibit multiple links of apoptosis pathway such as Caspase-3,Caspase-9,Caspase-10 and so on, combine with multiple cell cycleregulatory protein such as CyclinD, CyclinE to regulate mitosis. The latest studies confirm that SVV could express in nontumorous cells such as epithelial cells of gastric mucosa, kidney tubules and macrophage, and would not lead to cancer for it is not tumor specific genes.Therefore, this study cultivate rat aortic endothelial cells,over-expressed Survivin gene, to acquire an anti-apoptotic, proliferative vascular endothelial cell and discuss the mechanism of anti-apoptosis, proliferation, angiogenesis of SW to VECs, provide a certain theory and experiment basis for the treatment of PAD.PART I The Expression of Survivin in Rat Aortic EndotheIial CellsObjective:To discuss the expression of Survivin in rat aortic endothelial cell.Methods:We lysed rat aortic endothelial cell by eukaryotic cell lysis solution and then extract the protein. The expression of Survivin is detected through western-blot.Results:The expression rate of Survivin in normal cultured rat aortic endothelial cells is up to 100%. The outcomes of western-blot reveal that strips of Survivin is relatively light, grey level to 13.48, in rat aortic endothelial cells.Conclusion:Survivin express at a low level in rat aortic endothelial cells.PART ? The Effects of Survivin on angiogenesis i n RAECsObjective:To study the effects of Survivin on proliferation, anti-apoptosis, angiogenesis in rat aortic endothelial cell.Methods:We constructed the recombinant adenovirus, which contains the RNA of Survivin and transfected it into rat aortic endothelial cells. Then we detected Survivin gene expression after interference by RT-Qpcr and Western-blot. We detected the influence of Survivin on apoptosis, proliferation in vitro by flow cytometry and some related proteins in apoptosis pathway and cell cycle such as Caspase-3, bcl-2, CyclinD by Western-blot. We transplant RAEC, over-expressed Survivin, into nude mouse by intramuscular injection and detect RAEC, which express CD31 positive by immunohistochemistry.Results:Ad-Survivin/RNA displayed high transfection efficiency and over-expressed Survivin efficiency at MOI 300. Survivin over-expressed could inactivate or restrain the apoptosis passway by up-regulate bcl-2 and down-regulate Caspase-3 result in obvious decline of apoptosis in rat aortic endothelial cells. Cell cycle was significantly promoted by Survivin in vitro through up-regulation of CyclinD, which accelerates the transition from G1 phase to S phase. Immunohistochemical results reveal an obvious generation of new vessels in the intramuscular injection site two weeks after cell transplantation. It illustrates that the ability of anti-apoptosis, proliferation of Survivn promotes the process of angiogenesis and restrain the rejection reaction from allograft in vivo.Conclusion:RNAi of surviving would be a potential therapeutic approach for periphery artery disease. Survivin plays an important role in angiogenesis by a variety of molecular regulatory mechanisms.