马兜铃酸对人肾小管上皮细胞转分化和凋亡作用的体外实验研究

Aristolochic acid induced transdifferentiation and apoptosis in human tubular epithelial cells in vitro

目的 探讨马兜铃酸 (AA)对人类肾小管上皮细胞 (HKC)转分化和凋亡的影响。方法将不同浓度的AA(5、10、2 0和 4 0mg/L)分别加入HKC细胞培养液中培养 4 8h ,应用下列方法观察HKC细胞转分化 :间接免疫荧光法检测HKC细胞角蛋白和波形蛋白的表达 ,免疫组化双染色技术测定HKC细胞E 钙黏连蛋白和α 平滑肌肌动蛋白 (α SMA)的表达 ,流式细胞技术测定HKC细胞表达α SMA阳性百分率 ;应用Giemsa染色、TUNEL反应和琼脂糖凝胶电泳观察HKC细胞凋亡 ,流式细胞技术测定HKC细胞凋亡的百分率。结果 10mg/L的AA作用于HKC细胞 4 8h后角蛋白、E 钙黏连蛋白表达减弱 ,波形蛋白表达增强 ,HKC细胞表达α SMA阳性率 (14 .17± 0 .6 1) %比无血清对照组的 (3.5 7± 0 .5 2 ) %显著增高。 4 0mg/L的AA作用HKC细胞 4 8h后细胞凋亡 (5 3.4 % )比无血清对照组 (2 % )显著增高。 5和 2 0mg/L的AA未能引起HKC明显凋亡或转分化。结论 较低浓度 (10mg/L)的AA对HKC细胞有轻度的促转分化作用 ,较高浓度 (4 0mg/L)的AA作用 4 8h后引起多数HKC细胞凋亡 ,AA的上述作用可能与马兜铃酸肾病的发病机制有关。

Objective To examine the possible role of aristolochic acid (AA) in transdifferentiation and apoptisis of human tubular epithelial cell line (HKC) Methods Cultured HKC cells were divided into five groups: serum free (negative control) and treatment with AA at the concentrations of 5 mg/L, 10 mg/L, 20 mg/L and 40 mg/L for 48 hours, respectively. Transdifferentiation of HKC cells was observed with the following methods:detection of the expression of vimentin and cytokeratin of HKC cells with indirect immunoflourescence, determination of expression of E cadherin and α smooth muscle actin (α SMA) by indirect immunohistochemical double staining, and determination of the proportion of α SMA (+) HKC cells by flow cytometry. The apoptosis of HKC cells was observed with Giemsa staining, TUNEL reaction and agarose gel electrophoresis, and the ratio of apoptotic HKC cells was quantitatively analyzed by flow cytometry with propidium iodide staining Results The expression of cytokeratin and E cadherin reduced and that of vimentin increased in HKC cells treated with 10 mg/L of AA for 48 hours, and the expression of α SMA (+) in HKC cells treated with 10 mg/L of AA (14.17±0 61)% was significantly higher than that in serum free controls (3.57±0.52) %. Apoptosis of HKC cell treated with 40 mg/L of AA for 48 hours was 53.4%, significantly higher than that in serum free controls(2%). Treatment with 5 mg/L of AA and 20 mg/L of AA could not induce apoptosis and transdifferentiation of cells Conclusions Treatment with relatively low concentration of AA (10 mg/L) might induce slight transdifferentiation in cultured HKC cells and that with higher concentration of AA (40 mg/L) for 48 hours might induce apparent apoptosis of these cells, which suggested that transdifferentiation and apoptosis of tubular epithelial cells probably played important roles in aristolochic acid induced nephropathy.