马兜铃酸I诱导人肾小管上皮细胞转分化的作用及机制

TRANSDIFFERENTIATION OF CULTURED HUMAN RENAL TUBULAR EPITHELIAL CELLS INDUCED BY ARISTOLOCHIC ACID I

目的 :探讨马兜铃酸I(AristolochicacidI,AAI)在人类肾小管上皮细胞 (HKC)转分化中的可能作用 ,了解AAI引起肾小管间质损害的机制。 方法 :将体外培养HKC细胞分为无血清对照组 (C组 )和AAI实验组两组 ,在AAI组培养液中加入不同剂量AAI;两组HKC细胞分别培养 48h后 ,应用间接免疫荧光法检测HKC细胞角蛋白、波形蛋白和α 平滑肌肌动蛋白 (α smoothmuscleactin ,α SMA)表达的变化 ;应用流式细胞技术检测表达α SMA阳性(+)的HKC细胞百分率 ;采用免疫酶标技术 (ELISA)检测HKC细胞上清液中转化生长因子 β1 (TGFβ1 )的表达。 结果 :间接免疫荧光法观察 ,可见不同剂量AAI处理后的HKC细胞表达角蛋白减弱 ,表达α SMA及波形蛋白增强 ;应用流式细胞术检测 ,发现不同剂量AAI(1 0 ,2 0 ,40 ,80 μg/L)处理后 ,表达α SMA(+)的HKC细胞百分率与AAI剂量呈正相关 (r =0 0 86 7,P <0 0 0 5 ) ;当AAI为 2 0 ,40 ,80 μg/L时 ,HKC细胞表达α SMA百分率为 8 6 % ,9 6 % ,1 3 4 % ,较C组 (平均值为 3 1 % )明显增高 (P <0 0 5 )。应用ELISA法观测AAI对HKC细胞分泌TGFβ1的影响 ,可见C组HKC细胞有基础水平的TGFβ1分泌 (2 1 7%ng/L) ;当予一定剂量的AAI(1 0 ,2 0 μg/L)刺激HKC细胞 2 4h后 ,细胞上清液中TGF?

OBJECTIVE To investigate the possible role of Aristolochic acid I(AAI) in the phenotypic transformation of cultured human renal tubular epithelial cells line(HKC) METHODOLOGY The HKC cells were divided into two groups:serum free control group and AAI treatment groups with 12 48 hours’ treatment of AAI at different dosages. HKC were analyzed by immunohistochemistry, with monoantibodies to α smooth muscle actin(α SMA),vimentin and cytocratin respectively. The expression of α SMAof HKC cells was assessed by flow cytometry. The expression of transforming growth factor β(TGF β 1) in the supernatant of HKC cells was assessed with ELISA. RESULTS The expressions of vimentin and cytocratin were decreased in AAI treated HKC cells, while α SMA expression was increased by AAI in a dose dependant manner( r =0 0867, P <0 005).The basic expression of TGF β1 by HKC cells was 21 7 ng/L. The expression of TGF β1 in the supernatant of HKC cells increased significantly when cultured with AAI(10,20 μg/L)(75 6,65 6 ng/L vs 21 7ng/L, P <0 005).However, when the HKC cells were cultured with AAI at 40 or 80 μg/L,the TGF β1 levels from the supernatants were markedly decreased. CONCLUSION The transdifferentiation of HKC may be induced by AAI in vitro. AAI may have a diphasic role on TGF β1 expression in HKC cells. AAI induced transdifferentiation my be partially related to the increased TGF β1 secretion.