携带bcl-2基因慢病毒感染人原代卵巢颗粒细胞的凋亡  

Apoptosis of human primary ovarian granulose cells infected with lentivirus carrying bcl-2 gene

作　　者：王雪峰[1] 谭峰[1] 陈燕英[1] 刘木彪[1] 何援利[1] 

机构地区：[1]南方医科大学附属珠江医院妇产科,广东省广州市510282

出　　处：《中国组织工程研究》2013年第28期5209-5215,共7页Chinese Journal of Tissue Engineering Research

基　　金：2010年国家自然科学基金(81041101);2010年广东省自然科学基金(10451051501004704);2011年广州市科技计划项目(2011J4100087)~~

摘　　要：背景:慢病毒可以感染分裂及非分裂细胞,对于较难转染的原代卵巢颗粒细胞,慢病毒能否成功进行感染?目的:探讨携带bcl-2基因的慢病毒感染原代人卵巢颗粒细胞的效率及对细胞凋亡的影响。方法:利用分子生物学技术,构建携带bcl-2基因慢病毒载体,包装成高滴度慢病毒,将重组慢病毒在体外分别以不同感染复数(10,50,100,200,400)感染人卵巢原代颗粒细胞,感染24,48,72,96h后观察感染效率及细胞增殖情况,同时采用流式细胞仪检测细胞凋亡情况,利用反转录聚合酶链反应检测bcl-2基因的转录情况。结果与结论:原代培养人卵巢颗粒细胞24h即贴壁,集落样生长,呈多角形或梭形;当感染复数为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,其在颗粒细胞中的阳性率达60%;携带bcl-2基因的慢病毒感染原代培养的人卵巢颗粒细胞,可以过度分泌Bcl-2蛋白,能抑制卵巢颗粒细胞的凋亡。METHODS： The lentiviral vector carrying bcl-2 gene was constructed using molecular biology, and packaged into lentivirus with high titer. The resulting recombinant lentivirus carrying bc/-2 genes were then used to infect primary human ovarian granulosa cells in vitro at different multiplicity of infection, 10, 50, 100, 200, and 400. Infection efficiency and cell proliferation were observed at 24, 48, 72, and 96 hours following infection. Cell apoptosis was detected by flow cytometry, and bcl-2 gene transcription was assessed using reverse transcription PCR. RESULTS AND CONCLUSION： Primary human ovarian granulosa cells adhered at 24 hours, and exhibited polygon- or fusiform-shape and colony-like growth. When multiplicity of infection was 100, cell appearance and growth remained unchanged, and infection efficiency was high, which reached the peak up to 72 hours. Moreover the positive rate was up to 60% in granulosa cells. Lentivirus carrying bc/-2 gene could increase expression of Bcl-2 protein and inhibit apoptosis of primary ovarian granulosa cells.

关 键 词：组织构建 组织构建细胞学实验 BCL-2基因 慢病毒 卵巢颗粒细胞 细胞凋亡 基因转染 载体构建 卵巢早衰 增强型绿色荧光蛋白基因 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]