摘要
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.
出处
《基础医学与临床》
CSCD
北大核心
2007年第6期635-642,共8页
Basic and Clinical Medicine
基金
北京市自然科学基金(7042043)