摘要
目的 以体外培养的人近端肾小管上皮细胞系(HK-2)为研究对象，探讨刺激物马兜铃酸-I被洗脱(aristolochic acid-I，AA-I)后对HK-2细胞增殖抑制作用的影响。方法 以AA-I分别刺激细胞12、24、48h后去除刺激物，加入Hank’s液对细胞进行重复3次洗涤，继续培养24h后测定各项指标。台盘兰染色后细胞计数法观察存活细胞增殖反应；流式细胞仪分析细胞周期变化情况。结果 应用AA-I(10μg/ml)刺激HK-2细胞后，随着刺激时间的延长存活细胞数量逐渐减少,12、24、48h后存活细胞计数较对照组分别下降8%、25%和57%；细胞周期测定结果显示AA-I刺激HK-2细胞后，G2/M期细胞比例逐渐增高，到24h后G2/M期比例达对照组的1.28倍，48h后达正常对照组的3.8倍。以上情况在洗脱以后并没有得到改善，表现为洗脱后的12、24、48h组细胞数较相应时间点的刺激组分别下降45%、32%和30%；AA的G2/M阻滞作用在洗脱以后也没有好转，洗脱24h组G2/M比例达AA24h组的2.91倍，而与AA48h组差异无显著性。结论 AA-I(10μg/ml) 能够显著抑制HK-2细胞的增殖，表现为细胞数目减少，细胞周期阻滞于G2/M期。洗脱细胞外的AA-I后并未能改善AA-I刺激导致的细胞增殖抑制作用，提示细胞内蓄积的AA-I或其代谢物可能是导致细胞持续损伤的主要原因。
Abstract
Purpose After the cultured human renal tubular epithelial cells (HK-2 cells) were stimulated with aristolochic acid-1 (AA-I) for a period of 12-48 hours, whether removal of AA-1 in culture media can lessen the proliferation inhibition effect of AA-1. Methods HK-2 cells were cultured and stimulated with AA-1 for 12, 24 or 48 hours, washed with Hank抯 solution for 3 times, and then cultured in the medium free of AA-1 for 24 hours. Proliferation of the living cells was measured by the trypan blue method, and cell cycle was assessed by flow cytometry. Results The living cell number decreased by 8%, 25% and 57%, after the HK-2 cells were stimulated with AA-1(10μg/ml) for 12, 24 and 48 hours, respectively. AA-1 stimulated HK-2 cells showed higher proportion of G2/M stage, which increased to 1.28 and 3.8 times of the normal controls in those stimulated for 24 and 48 hours, respectively.Removal of AA-1 in the media did not improve the above changes. Of the HK-2 cells stimulated with AA-1 for 12, 24 and 48 hours and then removed the media AA-1, cell number deceased further by 45%, 32% and 30%, respectively. Of the HK-2 cells stimulated with AA-1 for 24 hours and then removed the media AA-1, the proportion of G2/M stage increased further by 2.91 times, equal to the value of the cells stimulated with AA-1 for 48 hours. Conclusion AA-1 (10μg/ml) inhibits the proliferation of HK-2 cells considerably, manifested by the decrease of living cell number and the blockage of cell cycle at G2/M stage. Removal of the extracellular AA-1 can not reverse the AA-1 induced cell proliferation inhibition, indicating that the intracellular accumulated AA-1 and its metabolites may be the important factors for the sustained injury of the cells.
关键词
马兜铃酸-I /
肾小管上皮细胞 /
细胞增殖 /
细胞周期
Key words
Renal tubular epithelial cell /
Cell proliferation /
Cell cycle
周 娜? 杨 莉 商 朴 唐嘉薇 王 璇 李晓玫.
刺激物洗脱对马兜铃酸-I所致肾小管上皮细胞增殖抑制作用的影响[J]. 中国血液净化. 2007, 6(8): 431-434
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