GPS2基因敲除小鼠胚胎成纤维细胞的永生化及其增殖凋亡的检测  

Immortalization of GPS2-deficient MEFs and detection of their proliferation and apoptosis ability

作　　者：田欢欢[1] 任广明[2] 詹轶群[2] 杨晓明[1,2] 尹荣华[2] 

机构地区：[1]安徽医科大学,合肥230032 [2]北京放射与辐射医学研究所,北京100850

出　　处：《军事医学》2016年第5期361-365,共5页Military Medical Sciences

基　　金：国家重大科学研究计划资助项目(2013CB910801)

摘　　要：目的利用SV40大T抗原(SV40LT)过表达慢病毒建立永生化的GPS2野生与敲除的小鼠胚胎成纤维细胞(MEF)系,并检测GPS2敲除对MEF增殖及凋亡的影响。方法构建p CDH-Flag-SV40LT-GFP慢病毒表达载体,并进行病毒包装。分离胚胎发育9.5 d(E9.5d)的MEF细胞,感染SV40LT慢病毒,连续传代培养50代以上,把仍存活且状态良好的GFP阳性细胞视作永生化成功的MEF细胞。利用高内涵细胞成像分析仪检测永生化MEF细胞的增殖情况,采用AnnexinⅤ/PI双染色、流式细胞仪检测细胞凋亡。结果成功构建p CDH-Flag-SV40LT-GFP慢病毒表达载体,获得滴度为4.03×108pfu/ml的病毒。分离E9.5d MEF细胞并感染上述病毒,阳性细胞扩大培养并稳定传代50代以上,成功建立了永生化的MEF细胞系。GPS2敲除MEF细胞的增殖能力明显低于野生型,而二者由于血清撤除所引起的凋亡并无明显差异。结论敲除GPS2抑制MEF细胞的增殖。Objective To immortalize the GPS2 wild-type and knockout mouse embryonic fibroblasts（ MEFs） with lentiviral particles expressing SV40 large T antigen（ SV40LT）,and to detect the proliferation and apoptosis of these immortalized MEFs. Methods For the lentivirus-mediated SV40 LT over-expression,full-length SV40 LT with a Flag-tag was cloned into the p CDH-MCS-T2A-cop GFP-MSCV vector to generate p CDH-Flag-SV40LT-GFP recombinant vector that was cotransfected with the packaging plasmids p MD2. G and ps PAX2 into HEK293 T cells to generate lentivirus. MEFs were isolated from fetus at embryonic day 9. 5（ E9. 5d）. GPS2 wild type and knockout MEFs were transfected with the lentivirus. The cells survived after more than 50 passages were deemed to be immortalized. The proliferation ability of the MEFs was measured while the apoptosis detection was submitted to Annexin Ⅴ / PI staining for FACS analysis. Results We constructed a p CDH-Flag-SV40LT-GFP vector and immortalized GPS2 wild type and knockout MEFs with lentiviral particles expressing SV40 LT. Slower proliferation and unchanged apoptosis of the immortalized GPS2 knockout MEFs were observed. Conclusion GPS2 is essential for the cell growth of MEFs and the deficiency of GPS2 suppresses the proliferation of MEFs.

关 键 词：小鼠 基因敲除 成纤维细胞 胚胎结构 SV40LT 永生化 细胞增殖 细胞凋亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]