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目的:观察马兜铃酸(aristolochic acid,AA)对体外培养的大鼠近端肾小管上皮细胞(NRK-52E)的损伤作用。方法:体外培养NRK-52E,分别以不同浓度的AA-Na刺激细胞,每组设6个复孔,孵育24 h。透射电镜观察NRK-52E细胞的超微结构,MTT法检测不同浓度AA对NRK-52E细胞增殖的影响,流式细胞仪检测NRK-52E细胞的凋亡,免疫细胞化学法检测AA对NRK-52E细胞α-平滑肌肌动蛋白(α-SMA)表达的影响;检测不同时间段(12、24、48 h)马兜铃酸对NRK-52E细胞转化生长因子-β1(TGF-β1)及内皮素-1(ET-1)mRNA和Ⅰ型胶原(ColⅠ)表达的影响。结果:①AA浓度低于10μg/ml时对NRK-52E的增殖无明显影响,20~40μg/ml浓度的AA可明显抑制NRK-52E细胞的增殖;②较高浓度的AA(40μg/ml)可明显刺激细胞凋亡;③AA5、10、20、40μg/ml均可刺激NRK-52E表达α-SMA,其中AA10μg/ml对α-SMA表达较强;④AA刺激NRK-52E细胞24 h后TGF-β1mRNA、ET-1 mRNA表达最多,48 h时表达水平下降,而COL-I的表达水平随时间的延长而上调,呈时间依赖性。结论:AA可直接损伤肾小管上皮细胞,促进其表达肌成纤维细胞标志蛋白α-SMA,即有促转分化作用,且其对NRK-52E细胞的损伤作用呈剂量及时间依赖性。
Objective: To observe the effect of aristolochic acid (AA) on the injury of rat proximal tubular epithelial cells (NRK-52E) in vitro. METHODS: NRK-52E was cultured in vitro and cells were stimulated with different concentrations of AA-Na, and 6 replicates in each group were incubated for 24 h. The ultrastructure of NRK-52E cells was observed by transmission electron microscope. The proliferation of NRK-52E cells was detected by MTT assay. The apoptosis of NRK-52E cells was detected by flow cytometry. The ARK was detected by immunocytochemistry. Effects of α-SMA expression; detection of transforming growth factor-β1 (TGF-β1) and endothelin in ARKN-52E cells at different time intervals (12, 24, 48 h) Effects of -1 (ET-1) mRNA and Type I Collagen (ColI) Expression. RESULTS: The concentration of AA below 10μg/ml had no effect on the proliferation of NRK-52E. The concentration of AA at 20-40μg/ml could significantly inhibit the proliferation of NRK-52E cells. 2The higher concentration of AA (40μg/ml) could be used. Apoptosis was significantly stimulated; 3,5,10,20,40 μg/ml of 3AA could stimulate the expression of α-SMA in NRK-52E, in which AA10μg/ml had strong expression ofα-SMA;4AA stimulated TGF-β1mRNA in NRK-52E cells after24h The expression of ET-1 mRNA was highest, and the expression level decreased at 48 h, while the expression level of COL-I was up-regulated with time, showing a time-dependent manner. Conclusion: AA can directly damage renal tubular epithelial cells and promote its expression of myofibroblast marker protein α-SMA, that is, it can promote transdifferentiation, and its damage to NRK-52E cells is dose- and time-dependent.