雷帕霉素对人肾小管上皮-肌成纤维细胞转化的抑制作用

Inhibitory effect of rapamycin on human renal tubular epithelial-myofibroblast transition

目的探讨雷帕霉素(Rapamycin,RAPA)对体外培养的人肾小管上皮细胞发生上皮肌成纤维细胞转化(Epi-thelial-Myofibroblast Transition,EMT)的作用,以及该作用与上皮细胞中锌指蛋白(Snail)基因水平变化的关系。方法体外培养的人近端肾小管上皮细胞(HKC),分为阴性对照组、转化生长因子β-1(TGF-β1)(1μg/L)阳性对照组和TGF-β1+RAPA组。TGF-β1+RAPA组不同浓度的雷帕霉素(0.1μg/L,1μg/L,10μg/L,100μg/L)与TGF-β1(1μg/L)共同作用,各组作用时间均为48h。用间接免疫荧光双染、RT-PCR、Western Blot方法分别检测细胞平滑肌细胞肌动蛋白(α-SMA)、E-钙黏素(E-cadherin)表达,同时用RT-PCR方法检测细胞Snail mRNA水平的变化。再选取最大作用浓度的雷帕霉素作用不同时间(12h、24h、48h、72h),用RT-PCR、Western Blot方法检测α-SMA表达水平变化。结果间接免疫荧光双染、RT-PCR、Western Blot方法检测均表明,TGF-β1阳性作用组较阴性对照组HKC细胞α-SMA mRNA及蛋白表达水平显著增强(P<0.05),而E-cadherin表达则几乎消失,Snail mRNA表达水平显著增强(P<0.05)。与阳性对照组相比,雷帕霉素(10μg/L,100μg/L)与TGF-β1共同作用组HKC细胞α-SMA mRNA及蛋白表达水平显著降低(P<0.05),而E-cadherin表达则有部分恢复,显著高于阳性对照组(P<0.05),而Snail mRNA表达水平比阳性对照组显著降低(P<0.05)。雷帕霉素以浓度依赖方式抑制TGF-β1诱导的HKC细胞Snail mRNA表达。结论应用体外培养的肾小管上皮细胞研究表明,雷帕霉素具有抑制肾小管上皮细胞EMT的作用。此作用可能与该药诱导的Snail表达下调有关。

Objective Rapamycin （RAPA） is an anti-proliferative immunosuppressant and has been used to suppress rejection of transplanted organs. In present study, we observed the effect of rapamycin on epithelial-myofibroblast transition （EMT）of cultured HKC cells in vitro. Methods Cultured human proximal tubular epithelial cells （HKCs） were divided into three groups： blank control, treated with TGF-β1 （1 μg/L） and treated with TGF-β1 （ 1 μg/L） plus rapamycin （0. 1, 1, 10, 100μg,/L）. The protein and mRNA for α-SMA and E-cadherin in HKC cells were determined by Western Blot and RT-PCR. The mRNA level of Snail in HKC was detected by RT-PCR. Results Rapamycin dramatically abrogated TGF-β1 induced α-SMA expression and restored E-cadherin expression in HKC cells in a dose-dependent manner. At a concentration of 100 μg/L, rapamycin almost completely blocked α-SMA mRNA and protein expression induced by TGF-β1 （ 1 μg/L）. Rapamycin also suppressed expression of α-SMA in HKC cells at both mRNA and protein level in a time dependent manner. We also found rapamycin dramatically abrogated TGF-β1 induced Snail mRNA expression in HKC cells in a doseependent manner. Conclusion Rapamycin may inhibit EMT of tubular cells in vitro. The downregulation of Snail expression might be one of the mechanisms of rapamycin blocking EMT.