骨形成蛋白7能减轻TGF-β1诱导的人足细胞损伤

Protective effect of bone morphogenetic protein 7 on TGF-β1 induced epithelial-mesenchymal transition of podocyte

摘要:

目的：观察骨形成蛋白（bone morphogenie protein 7，BMP-7）抑制转化生长因子β1（transforming growth factor-β1，TGF-β1）诱导的人足细胞转分化作用及其机制研究。方法：构建及鉴定pcDNA 3.1rh BMP-7质粒，体外培养人足细胞，先后予10、20、50、100、200 μg/mL重组的BMP-7诱导足细胞，分别在培养24、48、72 h收集细胞，流式细胞试验检测细胞凋亡率，以此选择重组BMP-7最适的刺激浓度和最适的作用时间点。实验分为正常对照组、TGF-β诱导组（TGF-β）及质粒转染组（PEGF-BMP-7），质粒转染组加入重组BMP-7 100 μg/mL预处理48 h后，而TGF-β诱导组和PEGF-BMP-7组分别予5 ng/mL TGF-β1处理足细胞后，通过免疫荧光观察足细胞Podocin、α-SMA、VIM的表达变化；收集细胞抽取总RNA和总蛋白，应用RT-PCR和Western blot观察重组BMP-7对人足细胞Podocin、α-SMA、VIM的mRNA和蛋白表达的影响。结果：①本实验成功构建pcDNA 3.1rh BMP-7质粒，转染的人足细胞能稳定表达BMP-7，质粒转染组BMP-7蛋白表达水平（0.487±0.012）较正常对照组（0.251±0.012）和TGF-β诱导组（0.151±0.015）明显增高（P＜0.001）；②Real-time PCR检测结果显示TGF-β诱导组足细胞Podocin的mRNA表达水平（0.285±0.013）较正常对照组（1.057±0.090）明显降低（P＜0.001），而质粒转染组足细胞Podocin的mRNA表达水平（0.693±0.077）较TGF-β诱导组有升高趋势（P=0.003）；TGF-β诱导组和质粒转染组足细胞的α-SMA（1.257±0.039和1.028±0.093）、VIM（1.114±0.097和0.821±0.059）的mRNA表达较正常对照组（0.357±0.089和0.403±0.020）明显升高（P＜0.001），质粒转染组足细胞α-SMA和VIM的mRNA较TGF-β诱导组有下降趋势（P=0.011，P=0.002）。Western blot检测结果显示TGF-β诱导组足细胞Podocin的蛋白表达水平（32.923±5.301）较正常对照组（101.807±9.208）明显降低（P＜0.001），而质粒转染组足细胞Podocin的蛋白表达水平（90.507±7.810）较TGF-β诱导组有升高趋势（P＜0.001）；TGF-β诱导组和质粒转染组足细胞的α-SMA（116.120±8.300和93.832±10.602）、VIM（124.016±9.702和100.801±6.801）的蛋白表达较正常对照组（35.730±4.892和43.801±2.600）明显升高（P＜0.001），质粒转染组足细胞α-SMA和VIM的蛋白较TGF-β诱导组有下降趋势（P=0.017，P=0.007）。结论：TGF-β1可诱导人足细胞发生转分化，导致其α-SMA、VIM表达增高，Podocin表达减少，BMP-7能显著抑制此现象，提示BMP-7可逆转足细胞EMT，对足细胞损伤具有保护作用。

Abstract:

Objective：To observe the protective effect of bone morphogenetic protein 7 on TGF-β1 induced epithelial-mesenchymal transition of podocyte. Methods：The pcDNA 3.1rh BMP-7 plasmid was constructed and identified. Human podocytes were cultured in vitro，with different concentrations（10，20，50，100，200 μg/mL） of recombinant BMP-7 stimulating podocytes for 24，48，72 h and were collected in the cell respectively. The death rates of cells were detected by flow cytometry，and the optimal stimulation concen－trations and time points of recombinant BMP-7 were selected. The experiment was divided into normal control group，TGF beta 1 stimulation group（TGF-β1） and plasmid transfection group（PEGF-BMP）. PEGF-BMP group was transfected podocyte with 100 μg/mL recombinant BMP-7 pretreatment after 48 hours；the human podocytes of TGF-β1 and PEGF-BMP groups were stimulated respectively with 5 ng/mL TGF-β1. Alterations of specific markers in cultured podocytes were observed by immunofluorescence；and 72 hours after treatment，the cells of different groups were harvested，the mRNA expressions of Podocin，SMA，VIM in podocyte were detected by RT-PCR and the protein expressions of Podocin，SMA，VIM in podocyte were detected by Western blot. Results：①pcDNA 3.1rh BMP-7 plasmid was constructed successfully and stably expressed BMP-7 was transfected in human podocytes. The expression level of BMP-7 protein in PEGF-BMP group（0.487±0.012） was significantly higher than that in normal control group（0.251±0.012） and TGF beta induction group（0.151±0.015）（P<0.001）. ②Real-time PCR analysis showed that mRNA expression of podocin was significantly decreased in TGF beta 1 group（0.285±0.013） than in normal control group （1.057±0.090）（P<0.001），while mRNA expression of podocyte was increased in PEGF-BMP-7 group（0.693±0.077） than in TGF beta 1 group（P=0.003）；the mRNA expression of α-SMA，VIM were significantly increased in TGF beta 1 group（1.257±0.039，1.114±0.097） and PEGF-BMP-7 group（1.028±0.093，0.821±0.059） than in normal control group（0.357±0.089，0.403±0.020）（P＜0.001），but mRNA expression of α-SMA，VIM were decreased in PEGF-BMP group than in TGF beta 1 group（P=0.011，P=0.002）. Western blot showed that protein expression of podocin was significantly decreased in TGF beta 1 group（32.923±5.301） than in normal control group（101.807±9.208）（P<0.001），while protein expression of podocyte was increased in PEGF-BMP-7 group（90.507±7.810） than in TGF beta 1 group（P＜0.001）；the protein expression of α-SMA，VIM were significantly increased in TGF beta 1 group（116.120±8.300，124.016±9.702） and PEGF-BMP group（93.832±10.602，100.801±6.801） than in normal control group（35.730±4.892，43.801±2.600）（P＜0.001），but protein expressions of α-SMA，VIM were decreased in PEGF-BMP-7 group than in TGF beta 1 group（P=0.017，P=0.007）. Conclusion：The expression of α-SMA and VIM in human podocyte induced by TGF beta 1 was in－creased，podocin expression decreased，while BMP-7 inhibited this effect and showed the protective effect on podocyte.