c-SKI对冠脉内皮细胞增殖及内皮-间充质转化的影响

目的:探讨核蛋白c-SKI对冠状动脉内皮细胞增殖能力及内皮-间充质转化的影响。方法:使用不同浓度的转化生长因子β1(TGF-β1)作用人冠脉内皮细胞不同时点,Western blot法检测各组细胞中c-SKI、间充质细胞标志物波形蛋白和α-平滑肌肌动蛋白(α-SMA)及内皮细胞标志物E-钙黏蛋白的表达。使用携带c-ski基因的慢病毒转染冠脉内皮细胞后采用RT-qPCR验证转染效率。将细胞分为4组:对照组、TGF-β1(5μg/L)组、c-ski基因感染+TGF-β1组及对照病毒感染+TGF-β1组。过表达c-SKI后,MTT实验和集落形成实验检测冠脉内皮细胞的增殖能力,Western blot检测波形蛋白、α-SMA、E-钙黏蛋白、Smad2、Smad3、p-Smad2和p-Smad3蛋白的水平。结果:TGF-β1处理冠脉内皮细胞后,c-SKI在冠脉内皮细胞中的表达呈剂量和时间依赖性下降(P<0.01)。MTT及集落形成实验结果显示,过表达c-SKI可显著抑制冠脉内皮细胞增殖(P<0.01)。Western blot检测结果显示,与病毒对照组相比,过表达c-SKI可显著下调冠脉内皮细胞中α-SMA和波形蛋白的表达量(P<0.01),上调E-钙黏蛋白的表达量(P<0.01),同时抑制Smad2和Smad3蛋白的磷酸化(P<0.01),逆转TGF-β1诱导的内皮-间充质转化。结论:内皮-间充质转化过程中c-SKI表达下调,而过表达c-SKI能够抑制冠脉内皮细胞增殖及内皮-间充质转化,其机制可能与调控TGF-β1/Smad信号通路活性有关。

Effect of c-SKI on coronary endothelial cell proliferation and endothelial-mesenchymal transition

AIM:To investigate the effect of cellular Sloan-Kettering Institute(c-SKI)on the proliferation and endothelial-mesenchymal transition of human coronary artery endothelial cells(HCAECs).METHODS:HCAECs were treated with transforming growth factor-β1(TGF-β1)at varying concentrations for different time points.Western blot was used to test the expression of c-SKI and mesenchymal markers such asα-smooth muscle actin(α-SMA)and vimentin.Meanwhile,the endothelial marker E-cadherin was also detected.HCAECs were transfected with c-ski gene mediated by lentivirus(LV),the efficiency of LV-SKI transfection was detected by RT-qPCR.The HCAECs were divided into 4 groups:control group,TGF-β1(5μg/L)group,LV-SKI+TGF-β1 group,LV-NC+TGF-β1 group.The cell viability and colony formation were measured by MTT assay and colony formation assay.The protein levels of vimentin,α-SMA,E-cadherin,Smad2,Smad3,p-Smad2 and p-Smad3 were determined by Western blot.RESULTS:The expression of c-SKI was down-regulated in the HCAECs treated with TGF-β1(P<0.01).Over-expression of c-SKI inhibited the proliferation of HCAECs(P<0.01).Compared with LV-NC group,over-expression of c-SKI down-regulated the expression ofα-SMA and vimentin(P<0.01),up-regulated the expression of E-cadherin(P<0.01),and inhibited the protein phosphorylation of Smad2 and Smad3(P<0.01),reversed the endothelial-mesenchymal transition induced by TGF-β1.CONCLUSION:The expression of c-SKI in the HCAECs is down-regulated in the process of endothelial-mesenchymal transition.Over-expression of c-SKI inhibits proliferation and endothelial-mesenchymal transition of HCAECs,the mechanism may be related to regulation of the TGF-β1/Smad signaling pathway.