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

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pc DNA 3.1rh BMP-7质粒,体外培养人足细胞,先后予10、20、50、100、200μg/m L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 100μg/m L预处理48 h后,而TGF-β诱导组和PEGF-BMP-7组分别予5 ng/m L 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m RNA和蛋白表达的影响。结果:(1)本实验成功构建pc DNA 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);(2)Real-time PCR检测结果显示TGF-β诱导组足细胞Podocin的m RNA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m RNA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m RNA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m RNA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。

Objective:To observe the protective effect of bone morphogenetic protein 7 on TGF-β1 induced epithelial-mesenchymal transition of podocyte. Methods:The pc DNA 3.1 rh BMP-7 plasmid was constructed and identified. Human podocytes were cultured in vitro,with different concentrations(10,20,50,100,200 μg/m L) 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 concentrations 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/m L recombinant BMP-7 pretreatment after 48 hours;the human podocytes of TGF-β1 and PEGF-BMP groups were stimulated respectively with 5 ng/m L 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 m RNA 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:(1) pc DNA 3. 1 rh 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).(2)Real-time PCR analysis showed that m RNA 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 m RNA expression of podocyte was increased in PEGF-BMP-7 group(0.693±0.077)than in TGF beta 1 group(P=0.003);the m RNA 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 m RNA 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 increased,podocin expression decreased,while BMP-7 inhibited this effect and showed the protective effect on podocyte.