自噬在电离辐射诱导的小鼠精母细胞GC-2凋亡过程中的作用

目的 探讨自噬在电离辐射诱导小鼠精母细胞GC-2凋亡过程中的作用.方法 将小鼠精母细胞GC-2分为空白对照组和不同剂量(2、4和8 Gy)60Co辐射处理组.采用原位末端转移酶标记法(TUNEL法)和流式细胞术检测细胞凋亡情况,通过蛋白质印迹实验观察自噬相关蛋白LC3(LC3-Ⅰ、LC3-Ⅱ)和Beclin1表达水平的改变,使用荧光显微镜观察C,C-2细胞内自噬小体的变化.用5mmol/L自噬抑制剂3-甲基腺嘌呤(3-MA)处理GC-2细胞2h再给予60Co辐射处理,观察自噬抑制剂联合电离辐射对细胞凋亡率的影响以及自噬相关蛋白表达水平的改变.结果 与空白对照组相比,GC-2细胞经60Co辐射处理后细胞凋亡率升高(P＜0.05),荧光显微镜下可见细胞自噬体明显增多,自噬相关蛋白LC3-Ⅱ以及Beclin1蛋白表达水平增加(P＜0.05).预先用5mmol/L 3-MA处理GC-2细胞2h再给予60Co辐射处理,自噬相关蛋白LC3-Ⅱ以及Beclin1蛋白表达水平与未经3-MA处理组相比降低(P＜0.05),细胞凋亡率升高(P＜0.05).结论 电离辐射可以诱导精母细胞发生自噬,抑制细胞自噬后可增强电离辐射对精母细胞的杀伤作用.

Role of autophagy in apoptosis of mouse GC-2 spermatocytes induced by ionizing radiation

Objective To investigate the role of autophagy in the apoptosis of GC-2 spermatocytes in mouse induced by ionizing radiation.Methods The mouse spermatocytes GC-2 cells were divided into control group and 2,4 and 8 Gy 60Co irradiation treatment groups The cell apoptosis was detected by in situ terminal transferase labeling (TUNEL) method and flow cytometry,the changes of autophagosome in GC-2 cells was observed by fluorescence microscope,and the expressions of autophagy-related proteins LC3 (LC3-Ⅰ and LC3-Ⅱ) and Beclin1 in GC-2 ceils were determined by Western blotting analysis.After treatment with autophagy inhibitor 3-methyladenine (3-MA) 2 h before ionizing radiation treatment,the effect of autophagy inhibitor combined with ionizing radiation on cell viability and the changes of autophagy-related protein expressions in GC-2 cells were observed.Results Compared with the control group,the apoptosis rote and the expression of LC3-Ⅱ and Beclin1 protdn of GC-2 cells in the irradiation treatment group were significantly increased (P＜0.05).Fluorescence microscopy showed that the cell autophagosome was increased.The expression of Beclin1 and LC3-Ⅱ protein in GC-2 cells treated with 5 mmol/L 3-MA was significantly lower than that in the 3-MA-untreated group (P＜0.05),and the apoptotic rate was significantly increased (P＜0.05).Conclusion Ionizing radiation can induce autophagy of spermatocytes,and the inhibition of autophagy can enhance the killing effect of ionizing radiation on spermatocytes.