论文部分内容阅读
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8Gy)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GC-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60 Co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。
Objective To investigate the role of autophagy in the apoptosis of mouse spermatocytes GC-2 induced by ionizing radiation. Methods The mouse spermatocyte GC-2 was divided into blank control group and different dose (2, 4 and 8Gy) 60Co radiation group. Cell apoptosis was detected by TUNEL and flow cytometry. Western blotting was used to observe the changes of the expression of autophagy-related proteins LC3 (LC3-Ⅰ, LC3-Ⅱ) and Beclin1 Fluorescence microscopy was used to observe the changes of autophagy in GC-2 cells. GC-2 cells were treated with 5 mmol / L autophagy inhibitor 3-methyladenine (3-MA) for 2 hours and then treated with 60Co radiation to observe the effect of autophagy inhibitor combined with ionizing radiation on apoptosis rate and autophagy Related protein expression levels change. Results Compared with the blank control group, the apoptosis rate of GC-2 cells after 60Co irradiation was increased (P <0.05). The number of autophagic cells was significantly increased under fluorescence microscope. The expressions of autophagy-related protein LC3-Ⅱ and Beclin1 protein The expression level increased (P <0.05). GC-2 cells were pretreated with 5mmol / L 3-MA for 2h and then irradiated with 60Co. The expressions of autophagy-related proteins LC3-Ⅱand Beclin1 were decreased compared with those without 3-MA treatment (P <0.05) The apoptosis rate increased (P <0.05). Conclusion Ionizing radiation can induce autophagy in spermatocytes, which can enhance the killing effect of ionizing radiation on spermatocytes after inhibiting autophagy.