论文部分内容阅读
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 construct immortalized mouse embryonic fibroblast (MEF) line of GPS2 wild-type and knockout using SV40 large T antigen (SV40LT) overexpression lentivirus and to detect the effect of GPS2 knockdown on MEF proliferation and apoptosis. Methods The pCDH-Flag-SV40LT-GFP lentiviral vector was constructed and virus-encapsulated. MEF cells of embryonic development 9.5 days (E9.5d) were isolated and infected with SV40LT lentivirus. The cells were subcultured for more than 50 passages. The surviving and good GFP positive cells were regarded as immortalized MEF cells. Proliferation of immortalized MEF cells was detected by high-content cell imaging analyzer. Apoptosis was detected by Annexin V / PI double staining and flow cytometry. Results The pCDH-Flag-SV40LT-GFP lentiviral vector was successfully constructed and a titer of 4.03 × 108pfu / ml was obtained. E9.5d MEF cells were isolated and infected with the above viruses. The positive cells were expanded and stably passaged for more than 50 passages, and the immortalized MEF cell line was successfully established. The proliferation ability of GPS2 knockout MEF cells was significantly lower than that of wild type, but there was no significant difference between the two groups due to the apoptosis induced by serum removal. Conclusion Knockdown of GPS2 inhibits MEF cell proliferation.