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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。
Objective To investigate the effect of aristolochic acid-I (AA-I) on the expression of HK-2 in human proximal tubular epithelial cell line (HK-2) cultured in vitro. Effects of proliferation inhibition. Methods The cells were stimulated with AA-I for 12, 24, and 48 hours, respectively. The stimuli were removed and the cells were washed with Hank’s solution for 3 times. After 24 hours of incubation, the indexes were measured. Cell proliferation assay was used to observe the cell viability after stained with cell suspension and the cell cycle was analyzed by flow cytometry. Results After HK-2 cells were stimulated with AA-I (10μg / ml), the number of viable cells decreased gradually with the stimulation time prolonged. The number of viable cells decreased by 8%, 25% and 57% %. The results of cell cycle assay showed that the proportion of cells in G2 / M phase increased gradually after HK-2 cells were stimulated by AA-I, reaching 1.28 times of G2 / M phase after 24 hours and 3.8 times that of normal control after 48 hours . The above conditions did not improve after elution. The number of cells in 12, 24 and 48h after elution were decreased by 45%, 32% and 30% respectively compared with the corresponding time points. G2 / M block in AA The effect did not improve after elution. The proportion of G2 / M in elution 24h group was 2.91 times that of AA24h group, but no significant difference with AA48h group. Conclusions AA-I (10μg / ml) can significantly inhibit the proliferation of HK-2 cells, showing a decrease in cell number and cell cycle arrest in G2 / M phase. Elution of extracellular AA-I did not improve AA-I stimulation-induced cell proliferation inhibition, suggesting that intracellular accumulation of AA-I or its metabolites may be the main cause of sustained cell damage.