| Cardiovascular disease has been a serious threat to human health.The main cause of it is coronary artery stenosis or obstruction causing insufficient blood supply and leading to heart dysfunction or disease.Clinical drug-eluting stents for treatment has been adopted,among whitch the most widely used is rapamycin-eluting stent applications.After eluting stents was implanted,rapamycin can inhibit smooth muscle cell proliferation and migration at the vascular injury spot,and prevent vascular restenosis and thrombus formation.Its potent immunosuppressive and anti-tumor effect is able to extend the lifespan of the graft and to prevent the anti-tumor potential risk due to the inhibition of the immune system.However,rapamycin releasing from eluting stent as the blood flows in the vessels,wether it has an impact on the intimal or no is currently rarely reported.This experiment is going to study the effects of rapamycin on human umbilical vein endothelial cells(ECV304),and to provide more more theoretical and experimental basis to evaluate rapamycin-eluting stents security.The experiment was divided into three groups,the negative control group,the administration groups of different concentrations of rapamycin and paclitaxel positive control group.First,using the MTT and SRB assay to detect rapamycin role in the proliferation of human umbilical vein endothelial cells;The fluorescence microscope was used to detect the effect of rapamycin on human umbilical vein endothelial cell morphology by HE33258 stain.The flow cytometry was used to explore the effect of rapamycin on the change of cell cycle in human umbilical vein endothelial cell.Confocal laser microscopy was used to study the effect of rapamycin on the change of Ca2+ concentration in human umbilical vein endothelial cell by Fluo-3/AM fluorescent probe.Western blot was used to explor the effects of rapamycin on Cyclin D1 and Cyclin A expression in ECV304 cells.Microplate reader was used to study the effects of rapamycin on mTOR content in ECV304 cells by ELISA.MTT results showed that Rapamycin can significantly inhibit the proliferation of ECV304 cells,and the effect was increased with the increased dose of rapamycin.SRB assay and MTT assay results were coincide.Fluorescence microscope viewd that rapamycin administration group compared with the negative control group the nucleus significantly increased,cell membrane integrity,it suggested that rapamycin may inhibit cell proliferation through the cell cycle.Flow cytometry analysed that rapamycin administration groups can significantly increased G1 phase peak,suggested it can block the cell cycle at G1 phase.Confocal laser scanning microscope viewd that rapamycin administration group compared with the negative control group can significantly increased Ca2+ concentration and and the effect was increased with the increased dose of rapamycin.Western Blot method results showed that rapamycin can significantly decrease Cyclin D1 and Cyclin A expression,and the negative contrast with significant difference(P<0.01).ELISA results showed that rapamycin can significantly improve mTOR content.The mode of action of rapamycin on mTOR is to inhibiti its phosphorylation,and making it exist with the form of mTOR molecular.The results suggested that rapamycin inhibit mTOR pathway by inhibiting mTOR molecule activated by phosphorylation.Conclusion:Rapamycin can inhibit the proliferation of human umbilical vein endothelial cells.It machanism is to inhibit mTOR phosphorylation activiting its pathway,and finally reduced the expression of Cyclin D1 and A.Blocking the cell cycle and inhibit cell proliferation.Further,cytosolic Ca2+ concentration increasing might also affect the physiological processes of the cell,inhibiting cell proliferation.Therefore,the rapamycin-eluting stent can also have an impact on undamaged parts of the intima of the blood vessels,suggesting that there is a potential risk of heart-eluting stents biological safety. |
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