SET蛋白对精原细胞株GC-1spg增殖和凋亡的影响

Effects of SET protein on proliferation and apoptosis of spermatogonia cell line GC-1 spg

目的探讨SET蛋白对精原细胞株GC-1spg增殖和凋亡的影响。方法使用含10%胎牛血清的DMEM高糖培养基在37℃、5%CO2条件下培养GC-1spg细胞,分对照组(转染无关序列腺病毒)和实验组[转染SET干涉腺病毒(AdH1-siRNA/SET)];GC-1spg接种于96孔板或者6孔板,转染腺病毒48h、72h后收集细胞,免疫荧光和共聚焦激光扫描显微镜检测SET蛋白在GC-1spg细胞中的表达与定位;提取细胞总蛋白,Western Blot检测转染前后SET蛋白的表达;细胞计数试剂盒-8(CCK8)和5-溴脱氧尿嘧啶核苷(BrdU)标记法检测细胞的数目和增殖情况;流式细胞仪检测细胞凋亡的变化。结果 SET蛋白表达于GC-1spg细胞的胞核和胞浆中,且胞浆分布多于胞核;转染SET干涉腺病毒后,干涉组GC-1spg细胞中的SET蛋白相对表达量为(0.217±0.044)显著低于对照组的(0.629±0.170)(P<0.05),同时GC-1spg细胞的数目减少,增殖减慢,且细胞凋亡率显著增加[干涉组(21.663±1.287)%,对照组(8.813±0.671)%](P均<0.01)。结论SET蛋白在精原细胞GC-1spg胞核和胞浆中均有表达,且胞浆分布多于胞核;GC-1spg细胞中SET蛋白表达降低,能够抑制细胞增殖、促进细胞凋亡,提示SET蛋白可能参与调节精子发生的过程。

Objective： To investigate the effects of SET protein on the proliferation and apoptosis of spermatogonia cell line GC-1 spg. Methods： GC-1 spg cells were cultured in DMEM/HIGH GLUCOSE with 10% fetal bovine serum in 5% COz at 37℃. The cells in control group were transfected with AdHI-siRNA/NS, and the cells in experimental group were transfected with AdHI-siRNA/SET. The cells were seeded in 96-well plate or 6- well plate and treated with adenovirus for 48 or 72 hours,and then the cells were collected. The expression and cellular location of SET protein were assessed by immunofluorescence and confocal laser scanning microscopy. The total proteins were extracted, and the expressions of SET protein before and after transfection were detected by Western blotting. Cell number and proliferation were tested by CCK8 assay and BrdU incorporation assay,while Cell apoptosis was measured by flow eytometry. Results： It was found that SET protein was expressed both in cytoplasm and nucleus of GC-1 spg cells,and it was mainly expressed in cytoplasm. The expression of SET protein in the GC-1 spg cells transfected with AdHI-siRNA/SET [（0. 217±0. 044）vs. （0. 629±0. 170）] was significantly lowered than that in control group（P〈0. 05）;while the apoptosis index was significantly increased [（21. 663± 1. 287） vs. （8. 813±0. 671）] （P〈0.01）,and the cell number and the proiiferation rate were decreased （P〈 0.01）. Conclusions： SET protein was expressed both in cytoplasm and nucleus of GC-1 spg cells,and it was mainly expressed in cytoplasm. Knockdown of SET protein inhibits proliferation,and promotes apoptosis of GC-1 spg cells,which suggests that SET protein may participate in the regulation of spermatogenesis.