甲基汞诱导大鼠皮层星形胶质细胞凋亡活性分析

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8 μmol·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≤0.1 μmol·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≥0.5 μmol·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≤0.1 μmol·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≥0.5 μmol·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 0.5 μmol·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 a strocyte. Results: Whit short-term (0-6 h)exposure to low doses of MMC (≤0.1 μmol·L^-1),the viability of astrocytes 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(≥0.5 μmol·L^-1),the astrocytes activity decreased with a concentration and time dependent manner(r_{concentration}=0.952-0.987,P< 0.05;r_time=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 (≥0.5 μmol·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 mitochondrial membrane potential (ΔΨ_m)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.