摘要
目的:研究马兜铃酸(AA)对人近端肾小管上皮细胞(HK-2)毒性作用中p53和Caspase-3活性的改变,对其在AA肾毒性作用中的意义进行探讨.方法:用乳酸脱氢酶(LDH)释放试验测AA对HK-2的直接毒性作用,相差显微镜观察细胞形态学改变,Hoechst33258染色法观察细胞凋亡形态的改变,流式细胞技术测细胞凋亡百分率,RT-PCR法测p53mRNA的表达水平,分光光度法测细胞Caspase-3活性改变.结果:AA浓度为80,160mg/L时,LDH释放率显著增高(P<0.01);10mg/LAA可诱导HK-2细胞凋亡百分率显著增高,AA为40mg/L时,细胞凋亡形态改变最明显和细胞凋亡比例最高(P<0.01);各组HK-2细胞p53mRNA表达水平有统计学差异;40mg/LAA诱导HK-2细胞凋亡时,细胞内Caspase-3活性显著升高(与正常组比较,P<0.01).结论:AA能诱导HK-2细胞凋亡,其凋亡途径可能是非p53依赖途径;凋亡过程中存在Caspase-3酶的激活.
AIM： To investigate the features of p53 expression and Caspase-3 activity assay in aristolochic acid （AA） induced renal tubular epithelial cell apoptosis. METHODS： The direct toxic effect of AA on HK-2 cells was evaluated by Lactate dehydrogenase（LDH） releasing essay. Apoptosis was observed under fluorescence microscope after the cells were stained by Hoechst 33258. The apoptosis rate of HK-2 cells induced by AA was estimated with flow cytometric analysis and the expression of wildtype p53 mRNA was measured by RT-PCR. Caspase-3 activity assay was performed by spectrophotometric method. RESULTS： The LDH release rate significantly increased after stimulation with AA at the concentrations of 80 and 160 mg/L（ compared with normal control, P 〈0.01 ）. AA（ at the concentrations of 20 and 40 mg/L） inhibited HK-2 cells growth and induced cell apoptosis. The apoptotic index in AA groups markedly increased compared with that in normal control（P 〈0.01 ）. The expressions of wildtype p53 mRNA in HK-2 cells after AA treatment（ at the concentrations of 10, 20 and 40 mg/L） were similar to those in control group. The activity of Caspase-3 markedly increased after AA treatment（at the concentrations of 20 and 40 mg/L） compared with that in normal control （P 〈 0.01 ）. CONCLUSION： AA is cytotoxie to renal tubular epithelial cells by inhibiting renal tubular epithelial cells growth and inducing apoptosis. Our results demonstrate that AA may induce apoptosis not through p53-independent pathway in renal tubular epithelial cells. Activation of Caspases-3 occurs in AA-induced apoptosis pathway.
出处
《第四军医大学学报》
北大核心
2009年第15期1398-1401,共4页
Journal of the Fourth Military Medical University
基金
广东省中医药局资助项目(104081)
作者简介
许勇芝．硕士，副教授．Tel：（0759）2387414Email：lxyzhi@126．com
