携带Bcl-2基因的慢病毒对原代培养的人卵巢颗粒细胞的影响

摘要

Objective To investigate the infectious effect of lentivirus carrying Bcl-2 gene on primary human ovarian granulosa cells (GCs) and its potentiality to induce apoptosis. Methods The lentivirus vector carrying Bcl-2 gene (pGC-FU-EGFP-bcl-2) was constructed and condensed to a high titer. The recombinant lentivirus with different MOI (10, 50, 100, 200, 400) were used to infect primary human ovarian GCs, and then the infection rate and cell proliferation of GCs were observed at 24, 48, 72 and 96 hours after infection. The GCs were incubated for 24 hours, and then divided into 3 groups: experimental group (GC-FU-Bcl-2 with MOI 100 was added into GCs), normal control group (only GCs), and empty vector control group (only the empty vector pGC-FU-EGFP was added into GCs). The expressions of Bcl-2 protein and mRNA in GCs were detected by Western blotting and RT-PCR, respectively, at the 3rd and 7th day after infection, and the apoptosis rate of GCs at day 3 was detected by flow cytometry Results The primary human ovarian GCs began to adhere with colony formation after 24h growth, and they were polygonal or spindle-shaped. When MOI was 100, the GCs maintained normal morphology and growth, and the infection rate in GCs reached the peak value (60%) after 72h cultivation. After being infected by the lentivirus carrying Bcl-2 gene, the expressions of Bcl-2 mRNA and protein could be detected in the GCs of experimental group, and the apoptotic rate of GCs was reduced significantly compared with that in empty vector control group (P<0.05)> Conclusion It seems that the lentivirus carrying Bcl-2 gene could increase the expression of Bcl-2 protein and, accordingly, inhibit the apoptosis of primary ovarian GCs.%目的 探讨携带Bcl-2基因的慢病毒对体外培养的原代人卵巢颗粒细胞感染效率及对细胞凋亡的影响.方法 构建携带Bcl-2基因的慢病毒载体,包装成高滴度慢病毒,将重组慢病毒在体外分别以不同感染复数(MOI)值(10、50、100、200、400)感染人卵巢原代颗粒细胞,观察感染24、48、72、96h后的感染效率及细胞增殖情况；将人卵巢颗粒培养24h后,分为3组.实验组:加MOI值为100的重组慢病毒GC-FU-Bcl-2；空白对照组:不加病毒；空载体对照组:加空载病毒GC-FU-EGFP.转染后第3、7天,采用Western blotting及反转录聚合酶链反应(RT-PCR)分别检测目的基因Bcl-2在人卵巢原代颗粒细胞中的蛋白及mRNA的表达水平.同时转染后第3天采用流式细胞仪检测细胞凋亡情况.结果 原代培养的人卵巢颗粒细胞24h即贴壁,集落样生长,呈多角形或梭形；当MOI为100时,细胞的形态和生长不受影响,且感染效率较高,感染后72h达高峰,感染率达60％.携带Bcl-2基因的慢病毒感染靶细胞后,实验组中检测到Bcl-2基因及蛋白的表达,且卵巢颗粒细胞的凋亡率明显低于空载体对照组.结论 携带Bcl-2基因的慢病毒感染原代培养的人卵巢颗粒细胞后可过度分泌Bcl-2蛋白,抑制细胞凋亡.