促肝细胞生长素部分逆转巨噬细胞趋化因子和马兜铃酸Ⅰ诱导的人肾小管上皮细胞转分化

Hepatocyte growth-promoting factor partially reverses monocyte chemotatic protein-1-and aristolochic acidⅠ-induced epithelial-to-mesenchymal transition of human kidney epithelial cells

目的观察促肝细胞生长素(hepatocyte growth-promoting factor,pHGF)对巨噬细胞趋化因子(monocyte chemo-tatic protein-1,MCP-1)协同马兜铃酸Ⅰ(aristolochic acidⅠ,AAⅠ)诱导的人肾小管上皮细胞(HKC)凋亡及上皮细胞-间质细胞转分化(epithelial-mesenchymal transition,EMT)的影响。方法体外培养的HKC随机分为:空白对照组、转分化模型组及不同浓度pHGF(0.15、1.5、15、150、1500ng/ml)处理组。转分化模型组采用MCP-1(0.1μg/ml)协同AAⅠ(10μg/ml)诱导HKC转分化模型;pHGF处理组采用不同浓度pHGF对转分化模型HKC进行处理;空白对照组常规培养。采用WST-8法和流式细胞术观察各组细胞增殖和凋亡情况;RT-PCR检测各组细胞α-SMA mRNA表达;免疫组化检测各组细胞α-SMA、TGF-β1、FN蛋白的表达。结果与空白对照组相比,转分化模型组、不同浓度pHGF处理组HKC细胞增殖抑制率,凋亡细胞所占比例,α-SMA mRNA表达均明显升高(P<0.01),提示转分化模型制备成功。与转分化模型组细胞相比,pHGF(150ng/ml)处理组HKC增殖抑制率明显降低(P<0.01),各浓度pHGF处理组HKC凋亡细胞所占比例均明显降低(P<0.01),HKC细胞α-SMA mRNA表达下调(150ng/ml pHGF处理组尤明显);α-SMA、TGF-β1、FN蛋白表达下调。结论pHGF(150ng/ml)可部分逆转MCP-1协同AAⅠ诱导的HKC增殖抑制、凋亡和EMT。

Objective To observe the influence of hepatocyte growth-promoting factor （ pHGF） on monocyte chemotatic protein-1-（MCP-1） and aristolochic acid Ⅰ（ AAⅠ）-induced epithelial-to-mesenchymal transition （EMT） and apoptosis of human kidney epithelial cell line（ HKC）. Methods The HKC cells were randomly divided into blank control group （ control groups） ,epithelial-to-mesenchymal transition model group （ model group） ,and pHGF inhibition group （ pHGF groups） with pHGF at different concentrations （ 0. 15,1. 5,15,150,and 1 500 ng/ml）. The EMT model was established by exposing HKC cells to MCP -1（ 0. 1 μg/ ml） and AAⅠ（ 10 μg/ml）. Cells in the pHGF groups were the model cells treated with different concentrations of pHGF. Cells in the control group were cultured routinely. WST -8 method and flow cytometry were used to observe the proliferation and apoptosis of HKC cells,respectively. The mRNA expression of α -smooth muscle actin（ α-SMA） was determined by reverse transcriptase-polymerase chain reaction（ RT-PCR） ,and the expression of α-SMA,fibronectin （ FN） ,and transforming growth factor-β1（ TGF-β1） in HKC cells were assessed by indirect enzyme immunohistochemistry. Results The cell inhibitory rate,apoptotic rate,and expression of α-SMA mRNA were significantly increased in the model group and pHGF groups compared with those in the control group （ P 〈 0. 01） ,indicating the successful establishment of EMT model. Compared with the model group,pHGF at 150 ng/ml,but not at other concentrations,significantly decreased the inhibition rate of HKC cells（ P 〈 0. 01）. The apoptotic rate of HKC cells in all the pHGF groups were significantly lower than that in the model group （ P 〈 0. 01）. pHGF at 150 ng/ml also greatly decreased the expression of α -SMA mRNA,and significantly down-regulated the expression of α-SMA,TGF-β1,and FN protein. Conclusion pHGF at 150 ng/ml can partly reverse MCP-1-and AA Ⅰ-induced HKC cell growth inhibition,apoptosis,and EMT.