mTOR-siRNA转染人晶状体上皮细胞对PI3K/AKT/mTOR信号通路蛋白p70S6K及4EBP1表达的影响

目的　研究mTOR-siRNA转染人晶状体上皮细胞系B3(human lens epithelial line B3,HLEB3)后，对PI3K/AKT/mTOR信号通路蛋白p70S6K及4EBP1表达的影响，并与雷帕霉素的作用作对比。方法　HLEB3分为mTOR-siRNA组（培养基加入Lipo2000和mTOR-siRNA）、control-siRNA组（培养基加入Lipo2000和control-siRNA）、Lipo2000组（培养基加入Lipo2000）、雷帕霉素组（培养基加入雷帕霉素）和空白对照组，于转染24 h、48 h、72 h后观察各组细胞的形态和密度；Western blot法检测各时间点各组细胞p70S6K及4EBP1蛋白的表达情况。结果　mTOR-siRNA组随着转染时间延长，HLEB3细胞分布稀疏、形态不规则，部分细胞贴壁不良。转染24 h、48 h、72 h时，mTOR-siRNA组细胞密度均较其他四组低，差异均有统计学意义（均为P<0.05）。转染24 h后，各组细胞4EBP1蛋白表达差异无统计学意义（P>0.05），但与空白对照组相比，mTOR-siRNA组及雷帕霉素组p70S6K蛋白表达均下降（均为P<0.05）；转染48 h、72 h后，与空白对照组相比，mTOR-siRNA组及雷帕霉素组4EBP1及P70S6K蛋白表达均下降（均为P<0.05），而control-siRNA组、Lipo2000组4EBP1、P70S6K蛋白表达与空白对照组相比差异均无统计学意义（均为P>0.05）。结论　mTOR-siRNA及雷帕霉素均可影响HLEB3的增殖及生长活性，并抑制PI3K/AKT/mTOR信号通路蛋白p70S6K和4EBP1的表达，且mTOR-siRNA的早期作用较雷帕霉素强。

The effect of mTOR-siRNA transfection of human lens epithelial cells on the expression of PI3K/AKT/mTOR signaling protein p70S6K and 4EBP1

Objective　To observe the effects of mTOR-siRNA transfection on expression of P70S6K and 4EBP1 protein of PI3K/AKT/mTOR signal transduction pathway in human lens epithelial line B3(HLEB3).Methods　The HLEB3 was divided into mTOR-siRNA group (lipo2000 and mTOR-siRNA were added in medium),control-siRNA group (Lipo2000 and control-siRNA were added in medium),and Lipo2000 group (Lipo2000 were added in medium),rapamycin group (rapamycin was added in medium) and blank control group.After transfection 24 h,48 h,72 h,the morphology and density of each group of cells were observed.The expression of P70S6K and 4EBP1 protein in each group at different periods was detected by Western blot.Results　The density of HLEB3 was sparse and irregular,and some cells were poorly attached as transfection time went by.After 24 h,48 h and 72 h transfection,the cell density of mTOR-siRNA group was lower than that of the other four groups,and the differences were statistically significant (all P＜0.05).After 24h transfection,there was no statistically significant difference in the expression of 4EBP1 protein in the five groups (P>0.05).However,compared with the blank control group,the expression levels of P70S6K protein in mTOR-siRNA group and rapamycin group were both decreased (all P＜0.05).After 48 h and 72 h transfection,compared with the blank control group,the expression levels of 4EBP1 and P70S6K protein in mTOR-siRNA group and rapamycin group were both decreased (P＜0.05),but the expression levels of 4EBP1 and P70S6K protein in control-siRNA group and Lipo2000 group were not statistically significant (all P>0.05).Conclusion　Both mTOR-siRNA and rapamycin can influence the proliferation and growth activity of HLEB3,and inhibit the expression of PI3K/AKT/mTOR signaling pathway protein P70S6K and 4EBP1,and the effect of mTOR-siRNA is stronger than that of rapamycin.