骨形成蛋白-7对肾小管上皮细胞转分化及结缔组织生长因子表达的影响

Effects of bone morphogenic protein-7 on transdifferentiation and the expression of connective tissue growth factor of human renal tubular epithelial cells induced by transforming growth factor-β1

目的观察骨形成蛋白-7（BMP-7）对转化生长因子-β1（TGF—β1）诱导人近端肾小管上皮细胞（HK-2）向间充质细胞转分化（EMT）及表达结缔组织生长因子（CTGF）的影响，探讨其抑制、逆转肾间质纤维化的可能机制。方法将体外培养的HK-2细胞分为对照组，3ng／ml TGF-β1组，TGF—β1同时加不同浓度BMP-7（100～400ng／ml）组。相差显微镜观察细胞形态改变；间接免疫荧光测定E-cadherin的表达；RT—PCR和Western印迹分别检测α-SMA、E—cadherin、CTGF mRNA和蛋白的表达。结果3ng／ml TGF—β1刺激48h后，HK-2细胞由立方形铺路石样转变为梭形长条样；去除TGF-β1，再加入200ng／ml BMP-7干预48h，可见到大部分细胞逆转回原来的形态。免疫荧光显示，E-cadherin蛋白在正常HK-2细胞质膜高表达；3ng／ml TGF—β1刺激后，E—cadherin表达明显减弱；去除TGF—β1，再加入200ng／ml BMP-7干预后，E—cadherin表达重新恢复。RT—PCR及Western印迹显示，3ng／ml TGF-β1刺激48h后，α—SMA mRNA及蛋白表达明显上调、E—cadherin mRNA及蛋白表达明显减少，同时CTGF mRNA及蛋白表达明显增加（P〈0．01）；BMP-7与TGF—β1共同刺激48h后，BMP-7呈剂量依赖性抑制TGF-β1，诱导下仪．SMA mRNA及蛋白的表达，并逐渐恢复E—cadherin mRNA及蛋白的表达，同时下调CTGF mRNA及蛋白的表达，400ng／ml BMP-7组与TGF-β1，组相比差异有统计学意义（P〈0．01）。结论BMP-7能阻断并逆转TGF—β1诱导的EMT，下调CTGF的表达，这可能是其逆转肾间质纤维化的重要作用机制。

Objective To investigate the effects of bone morphogenic protein （BMP）-7 upon epithelial-to-mesenchymal transition （EMT） and the expression of connective tissue growth factor （CTGF） in human renal proximal tubular epithelial cells （ HK-2 ） induced by transforming growth factor-β1 （ TGF-β1 ） and to explore the possible mechanisms of BMP-7 for the inhibition and reversal of renal interstitial fibrosis. Methods HK-2 cells were treated with TGF-β1 or a combination of TGF-β1 and BMP-7. RT-PCR and Western blot were used to determine the mRNA and protein expression of α-SMA, E-cadherin and CTGF. For EMT reversal experiments, when TGF-β1-induced EMT occurred, then the medium was removed and replaced with medium containing 200 ng/ml BMP-7. After 48 h, the morphological changes and the expression of E-cadherin were assessed by phase contrast microscopy or immunofluorescent microscopy. Results The control cells displayed typical cobblestone morphology of epithelial cells. 3 ng/ml TGF-β1 induced profound morphologic changes after 48 h, with cells becoming elongated in shape, but addition of 200 ng/ml BMP-7 for 48 h restored the epithelial morphology of HK-2 cells. Indirect immunofluorescence showed that treatment of 3 ng/ml TGF-β1 resulted in a distinct loss of E-cadherin staining in the plasma membrane of HK-2 cells but subsequent treatment with 200 ng/ml BMP-7 largely restored the E-cadherin protein staining. 3 ng/ml TGF-β1 significantly up-regulated the mRNA and protein expression of α-SMA,reduced the expression of E-cadherin and increased the expression of CTGF after 48 h （ vs. control, P 〈 0. 01 ）. BMP-7 dramatically suppressed the mRNA and protein expression of α-SMA, restored the expression of E-eadherin and prevented the expression of CTGF in a dose-dependent manner after co-incubation with TGF-β1 for 48 h （400 ng/ml BMP-7 ＋ TGF-β1 vs. TGF-β1 alone, P 〈 0. 01 ）. Conclusions BMP-7 exerts its antifibrotic effect partially through blocking and reversing TGF-β1 -induced EMT and down- regulating the expression of CTGF in human renal tubular epithelial cells.