慢病毒介导的Bcl-2基因对磷酰胺氮芥诱导人原代卵巢颗粒细胞凋亡的保护作用  

Protective effect of lentivirus-mediated Bcl-2 gene transfection against phosphoramide mustard-induced apoptosis of human ovarian granulosa cells

作　　者：王雪峰[1] 何援利[1] 付霞霏[1] 彭冬先[1] 

机构地区：[1]南方医科大学珠江医院妇产科,广东广州510282

出　　处：《南方医科大学学报》2012年第7期932-936,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81041101);广东省自然科学基金(10451051501004704)~~

摘　　要：目的探讨携带Bcl-2基因的慢病毒对磷酰胺氮芥诱导体外培养的原代人卵巢颗粒细胞凋亡的保护作用。方法利用分子生物学技术,成功构建携带Bcl-2基因慢病毒载体,包装成高滴度慢病毒,将重组慢病毒体外感染人卵巢原代颗粒细胞,再加入浓度30μmol/L的磷酰胺氮芥诱导细胞凋亡。分为4组进行实验:(a)实验组:pGC-FU-Bcl-2+磷酰胺氮芥(PM);(b)空载对照组:pGC-FU-EGFP+PM;(c)实验对照组:只加PM;(d)空白对照组:不加PM及病毒。采用流式细胞仪、Hoechst 33258染色检测卵巢颗粒细胞凋亡情况;利用Western blot检测Bcl-2蛋白的表达情况。结果空白对照组(GCs),凋亡峰不明显,颗粒细胞凋亡率为(1.93±0.28)%,实验组(GCs+PM+pGC-FU-Bcl-2)出现微弱的凋亡峰,颗粒细胞凋亡率为(6.99±0.55)%,比实验对照组(GCs+PM)及空载对照组(GCs+PM+pGC-FU-EGFP)显著低,但显著高于空白对照组(P<0.05)。实验组Bcl-2蛋白的表达明显增高,显著高于其余各对照组(P<0.05)。结论携带Bcl-2基因的慢病毒感染靶细胞后可以过度分泌Bcl-2蛋白,显著抑制卵巢颗粒细胞的凋亡,保护PM对颗粒细胞的理化损伤。Objective To investigate the effect of lentivirus-mediated Bcl-2 gene transfection in protecting human primary ovarian granulosa ceils against phosphoramide mustard （PM）-induced apoptosis. Methods Granulosa cells were isolated from the follicle fluid of women undergoing in vitro fertilization and embryo transfer. The lentiviral vectors carrying Bcl-2 gene （pGC-FU-Bcl-2） and enhanced green fluorescence protein （pGC-FU-EGFP） were constructed and packaged into high-titer lentiviruses. The resulting recombinant lentivirus carrying Bcl-2 and EGFP genes or the empty vector were used to infect the primary human ovarian granulosa cells, followed by addition of PM in the cell culture, with untreated granulosa ceils as the control. The cell apoptosis was detected by Annexin V and Horchst 33258 staining, and the expression of Bcl-2 protein was assessed using Western blotting. Results The control granulosa cells showed an apoptotic rate of （1.93±0.28）%. The cells infected with pGC-FU-Bcl-2 prior to PM exposure had a apoptotic rate of （6.99±0.55）%, significantly higher than that of the control cells, but significantly lower than that of the cells with PM exposure only and those infected with the empty vector before PM exposure （P〈0.05）. The expression of Bcl-2 was the highest in the ceils infected with pGC-FU-Bcl-2 prior to PM exposure （P〈0.05）. Conclusion Lentivirus-mediated Bcl-2 gene transfection can protect human ovarian granulosa cells against PM-induced apoptosis by upregulating Bcl-2 protein expression.

关 键 词：BCL-2基因 慢病毒 化疗药物 细胞凋亡 原代卵巢颗粒细胞 

分 类 号：R363[医药卫生—病理学]