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目的:利用稳定过表达Ankyrin repeat domain 49(ANKRD49)的GC-1(小鼠精原细胞)细胞模型,探讨ANKRD49过表达对GC-1细胞凋亡的影响。方法:40J/m~2紫外线(UV)刺激GC-1细胞2 min诱导凋亡,分别利用流式细胞术、Hoechst33258染色和免疫印迹技术检测过表达ANKRD49对GC-1细胞线粒体膜电位、核浓缩情况和凋亡相关蛋白多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)、Cleaved-Caspase-3、Bcl-xL和Bax的表达变化,以反映ANKRD49对GC-1细胞凋亡的影响。结果:流式细胞术检测结果表明稳定过表达ANKRD49的GC-1细胞线粒体膜电位下降率和细胞凋亡率均明显低于空载体组和裸细胞组(P<0.05)。Hoechst33258染色结果显示ANKRD49稳定过表达组细胞的凋亡百分比明显低于其它组(P<0.05)。Western blotting检测结果显示ANKRD49稳定过表达组Cleaved-PARP、Cleaved-Caspase-3蛋白的表达水平明显低于对照组,而Bcl-xL蛋白表达水平明显高于对照组(P<0.05)。结论:ANKRD49过表达可抑制UV诱导的GC-1细胞凋亡,其作用可能是直接或间接通过促进Bcl-xL的表达而实现的。
OBJECTIVE: To investigate the effect of ANKRD49 overexpression on the apoptosis of GC-1 cells by GC-1 (mouse spermatogonial) cell model stably overexpressing Ankyrin repeat domain 49 (ANKRD49). Methods: The apoptosis of GC-1 cells was induced by 40J / m ~ 2 ultraviolet light (UV) for 2 min. Flow cytometry, Hoechst33258 staining and Western blotting were used to detect the effect of ANKRD49 on mitochondrial membrane potential, The changes of PARP, Cleaved-Caspase-3, Bcl-xL and Bax expression in the apoptosis-related protein were detected to reflect the effect of ANKRD49 on the apoptosis of GC-1 cells. Results: Flow cytometry results showed that mitochondrial membrane potential decline rate and apoptosis rate of GC-1 cells stably over-expressing ANKRD49 were significantly lower than those of empty vector group and naked cell group (P <0.05). Hoechst33258 staining showed that the percentage of apoptotic cells in ANKRD49 overexpressing cells was significantly lower than that in other groups (P <0.05). The results of Western blotting showed that the expression of Cleaved-PARP and Cleaved-Caspase-3 protein in ANKRD49 stable overexpression group was significantly lower than that in control group, while the expression of Bcl-xL protein was significantly higher than that in control group (P <0.05). CONCLUSION: Overexpression of ANKRD49 can inhibit UV-induced apoptosis of GC-1 cells and its effect may be directly or indirectly through the promotion of Bcl-xL expression.