TGF-β1体外诱导肝星形细胞活化中自噬和mTOR通路的变化

［摘要］目的: 观察体外转化生长因子 β1(transforming growth factor-beta1，TGF β1)诱导的肝星形细胞活化中自噬水平和哺乳动物西罗莫司靶蛋白(mammalian target of rapamycin，mTOR)通路相关分子表达的变化。方法: 体外TGF β1诱导人肝星形细胞株LX 2，分为对照组、诱导48 h和72 h组，分别常规培养，加入2 ng/mL TGF β1培养48 h和72 h。观察细胞形态改变，应用荧光定量PCR和蛋白质印迹法检测TGF β1、α 平滑肌肌动蛋白(α smooth muscle actin，α-SMA)和LC3BⅡ以及mTOR、磷酸化mTOR(p-mTOR)和核糖体蛋白S6激酶(p70 ribosomal protein S6 kinase，p70S6K)的表达。结果： TGF β1诱导48 h后，部分LX-2细胞变长呈纺锤形或梭形，诱导72 h后，90%以上LX 2细胞呈多突纺锤形，形态向成纤维细胞样细胞转变明显。与对照组相比，诱导48 h组细胞中α-SMA、TGF-β1和LC3BⅡ mRNA及蛋白表达升高(P<0.05)，诱导72 h组升高更明显(P<0.01)；诱导48 h组细胞中p mTOR/mTOR比值和p70S6K蛋白表达降低，诱导72 h组降低更明显(P<0.05) 。结论： TGF-β1体外诱导肝星形细胞活化中自噬蛋白表达增加，而mTOR表达受到抑制。

Changes of autophagy and mTOR pathway in the TGF-β1 induced activation of hepatic stellate cells in vitro

Abstract]Objective: To observe changes of autophagy and the expression of mammalian target of rapamycin(mTOR) signal pathway in hepatic stellate cells activated by transforming growth factor beta1(TGF β1). Methods: The human hepatic stellate cell line LX 2 induced by TGF β1 were divided into three groups, including control group, TGF β1 48 h group and 72 h group, which were incubated with routine medium, 2 ng/mL TGF β1 for 48 h and 72 h, respectively. The morphological features of LX 2 cells in three groups was observed. The mRNA expression of α smooth muscle actin(α SMA), TGF β1 and LC3BⅡ was detected by quantitative real time PCR. The protein expression of α SMA, TGF β1, LC3BⅡ, mTOR, phosphorylate mTOR(p mTOR) and p70 ribosomal protein S6 kinase(p70S6K) were detected by Western blotting. Results: Some of LX 2 cells activated by TGF β1 transformed into short spindle after 48 h treatment, while more than 90 percent of LX 2 cells activated by TGF β1 transformed into fibroblast after 72 h treatment. Compared with the control group, the mRNA and protein expression of α SMA, TGF β1 and LC3BⅡ increased in 48 h group(P<0.05), and increased more significantly in 72 h group (P<0.01); the ratio of p mTOR to mTOR and the protein expression of P70S6K decreased in 48 h group (P<0.05), and decreased more significantly in 72 h group(P<0.05). Conclusion: The expression levels of autophagy proteins in hepatic stellate cells induced by TGF β1 were increased, which might be attributed to inhibition of mTOR pathway.