甲基汞诱导大鼠皮层星形胶质细胞凋亡活性分析

Activity analysis of methylmercury-induced apoptosis of rat cortical astrocyte

目的：研究氯化甲基汞（MMC）对原代培养的大鼠皮质星形胶质细胞存活和凋亡的影响。方法：MMC 0.01、0.05、0.1、0.5、1、2、4和8μmol·L-1孵育原代培养大鼠皮质星形胶质细胞3∽4 h,MTT检测星形胶质细胞活性,Hoechst 33342荧光染色和流式细胞仪测定细胞的凋亡,JC-1荧光探针检测线粒体膜电位,Western-blotting法测定凋亡诱导因子和细胞色素C的释放。结果：MMC浓度≤0.1μmol·L-1作用0∽6 h,星形胶质细胞的活力无显著变化;暴露时间12∽24 h,细胞活力显著降低（P〈0.05）。MMC浓度≥0.5μmol·L-1,星形胶质细胞活力呈浓度依赖性和时间依赖性降低（r浓度=0.952∽0.987,P〈0.05;r时间=0.831∽0.976,P〈0.05）;MMC浓度≤0.1μmol·L-1,随着时间延长使星形胶质细胞凋亡率增加（P〈0.05）。MMC浓度≥0.5μmol·L-1作用12 h,凋亡率最高,12 h后凋亡率降低,坏死率增高（P〈0.05）;MMC 0.5μmol·L-1呈时间依赖性降低线粒体膜电位（r=0.988,P〈0.05）,凋亡诱导因子和细胞色素C的释放增加。结论：氯化甲基汞可通过线粒体凋亡途径诱导星形胶质细胞凋亡。

Objective：To investigate the effects of methylmercury on the survival and apoptosis of rat cortical astrocyte. Methods： Different concentration of methylmercury （0.01,0. 05,0. 1,0.5,1,2,4,8 μmol ·L-1 ）was used to treat astrocyte for 3-4 hours. Cell viability was evaluated via MTT, and astrocyte apoptosis was determined by Hoechst 33324 staining and flow cytometry apparatus. Astrocyte mitochondrial membrane potential was assessed with the fluorescent probe JC-1. Western blotting was employed for the analyses of AIF and cytochrome C level in astrocyte. Results：Whit short-term （0-6 h）exposure to low doses of MMC （P〈0. 1μmol·L-1） ,the viability of astro- cytes did not change significantly（P 〉0. 05）. But with prolonged exposure （12-24 h）to low doses of MMC,the viability of astrocytes was significantly reduced （P 〈 0. 05 ）. Whith exposure to high doses of MMC （ ≥0. 5μmol·L-1 ）, the astrocytes activity decreased with a concentration and time dependent manner （r tration = 0. 952 --0. 987, P 〈 0. 05 ; rtime = 0. 831 - 0. 976, P 〈 0. 05 ）. Low concentration of MMC （ ≤0. 1 μmol·L-1 ） increased the apoptosis rate of astrocytes along with the extension of exposure time （P 〈 0. 05 ）. High concentration of MMC （ ≥0. 5μmol·L-1 ）increased the apoptosis rate（P 〈0. 05）,and the highest apoptosis rate was observed at 12 hours;after 12 hours, the apoptosis rate decreased and necrosis rate increased （P 〈 0. 05 ）. MMC （0. 5 μmol·L-1） induced mito- chondrial membrane potential （ △ψm ） loss（ P 〈 0. 05 ） and subsequent release of pro-apoptotic factor cytochrome C and apoptosis-inducing factor （AIF）. Conclusion： Methylmercury can induce the apoptosis of cortical astrocyte via mitochondria apoptosis.