雷帕霉素靶蛋白信号通路介导转化生长因子-β2诱导的后发性白内障的分子机制研究

摘要

目的：探讨雷帕霉素靶蛋白（ mammalian target of rapamycin ， mTOR）信号通路与转化生长因子（ transforming growth factor ，TGF）-β2诱导的晶状体上皮细胞间质化的相关机制。方法显微镜观察TGF-β2诱导的晶状体上皮细胞的表型变化，Western blot进一步验证TGF-β2诱导的HLEB-3上皮间质转化模型。 MTT检测TGF-β2诱导上皮间质化后以及雷帕霉素对细胞增殖的影响。 Western blot在分子水平检测上皮间质化和mTOR信号通路之间的机制关联。结果加入TGF-β2诱导处理24 h后，用显微镜观察HLEB-3的细胞形态由椭圆形变为星形或纺锤形，细胞连接减少。 Western blot检测发现TGF-β2诱导后的HLEB-3中上皮细胞标志蛋白E-cadherin表达水平明显降低，而间质细胞标记蛋白α-SMA的表达水平显著降低，差异有统计学意义（P＜0．05）。 MTT检测发现雷帕霉素预处理可以抑制TGF-β2对上皮细胞增殖的促进作用。 Western blot显示当用雷帕霉素抑制mTOR信号通路的激活后，可以逆转TGF-β2对α-SMA的表达的上调作用，促进上皮细胞标志蛋白E-cadherin的表达。结论 mTOR信号通路介导TGF-β2诱导的上皮细胞间质化作用，促进后发性白内障的发生。%Objective To investigate the mechanism of the association between mammalian target of rapamycin ( mTOR) sig-nal pathway and TGF-β2-induced lens epithelial mesenchymal transition .Methods Phenotypic changes in lens epithelial cells induced by TGF-β2 were closely observed by microscopy .Western blotting was used to further verify the HLEB 3 epithelial mesenchymal transi-tion model induced by TGF-β2.MTT was applied to detect the effects of TGF-β2 after induction of epithelial mesenchymal and Rapamy-cin on cell proliferation .Western blotting was used to detect the mechanism of the relationship between epithelial mesenchymal transi -tion and mTOR signal pathway at the molecular level .Results After TGF-β2 treatment for 24 hours, microscopic observation revealed that HLEB-3 cells were transformed from the oval shape into the star or fusiform shape , and cell connection was also reduced .Western blotting revealed that the expression levels of epithelial protein marker E-cadherin in HLEB-3, following induction of TGF-β2, was obvi-ously decreased , while the expression levels of the stromal cell protein marker α-SMA were significantly decreased , and statistical sig-nificance could be noticed when comparisons were made between them (P<0.05).MTT test also indicated that Rapamycin pretreat-ment could inhibit the enhancing effect of TGF-β2 on epithelial cell proliferation .Western blotting showed that Rapamycin could reverse the up-regulation of α-SMA by TGF-β2 and promote the expression of epithelial protein marker E-cadherin, following the activation of mTOR signal pathway inhibited by Rapamycin .Conclusion Epithelial mesenchymal transition induced by mTOR signal pathway and mediated by TGF-β2 could promote the onset of after-cataract.