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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 transdifferentiation of human renal tubular epithelial cells (HKC) and to understand the mechanism of AAI-induced tubulointerstitial damage. Methods: HKCs were cultured in vitro and divided into two groups: serum-free control group (C group) and AAI test group. Different doses of AAI were added to the culture medium of AAI group; HKC cells of the two groups were cultured for 48 h and detected by indirect immunofluorescence assay. Changes in the expression of keratin, vimentin, and α smooth muscle actin (α SMA) in HKC; the percentage of HKC cells expressing α SMA positive (+) was detected by flow cytometry; immunolabeling technique (ELISA) was used. The expression of transforming growth factor β1 (TGFβ1) in HKC cell supernatants was examined. RESULTS: By indirect immunofluorescence assay, HKC cells treated with different doses of AAI showed decreased expression of keratin protein and increased expression of α SMA and vimentin. Flow cytometry was used to detect different doses of AAI (10, 20, 40 ). After 80 μg/L treatment, the percentage of HKC cells expressing α SMA(+) was positively correlated with the AAI dose (r = 0 086 7, P <0 0 05); when AAI was 2 0, 40, 80 μg/ At the time of L, the percentage of α-SMA expressed in HKC cells was 86%, 96%, and 134%, which was significantly higher than that in group C (average of 31%) (P < 0.05). The effect of AAI on the secretion of TGFβ1 in HKC cells was observed by ELISA. The results showed that basal levels of TGFβ1 secretion (2 17 ng/L) in HKC group C; when given a dose of AAI (10, 20 μg/L) After stimulating HKC cells 24 h, TGF in cell supernatants?