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  

Changes of aristolochic acid-1 induced proliferation inhibition after removal of the stimulant in renal tubular epithelial cell culture

作　　者：周娜[1] 杨莉[1] 商朴[2] 唐嘉薇[1] 王璇[2] 李晓玫[1] 

机构地区：[1]北京大学第一医院肾内科北京大学肾脏病研究所卫生部肾脏疾病重点实验室 [2]北京大学药学院,北京100083

出　　处：《中国血液净化》2007年第8期431-434,共4页Chinese Journal of Blood Purification

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。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 ofAA-1. Methods HK-2 cells were cultured and stimulated with AA-1 for 12, 24 or 48 hours, washed with Hank＇s solution for 3 times, and then cultured in the medium free ofAA-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 （10 μg/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 （10μ g/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 

分 类 号：R692.6[医药卫生—泌尿科学]