本文關(guān)鍵詞： 小鼠 基因敲除 成纖維細(xì)胞 胚胎結(jié)構(gòu) SVLT 永生化 細(xì)胞增殖 細(xì)胞凋亡　出處：《軍事醫(yī)學(xué)》2016年05期 　論文類型：期刊論文

【摘要】：目的利用SV40大T抗原(SV40LT)過表達(dá)慢病毒建立永生化的GPS2野生與敲除的小鼠胚胎成纖維細(xì)胞(MEF)系,并檢測(cè)GPS2敲除對(duì)MEF增殖及凋亡的影響。方法構(gòu)建p CDH-Flag-SV40LT-GFP慢病毒表達(dá)載體,并進(jìn)行病毒包裝。分離胚胎發(fā)育9.5 d(E9.5d)的MEF細(xì)胞,感染SV40LT慢病毒,連續(xù)傳代培養(yǎng)50代以上,把仍存活且狀態(tài)良好的GFP陽性細(xì)胞視作永生化成功的MEF細(xì)胞。利用高內(nèi)涵細(xì)胞成像分析儀檢測(cè)永生化MEF細(xì)胞的增殖情況,采用AnnexinⅤ/PI雙染色、流式細(xì)胞儀檢測(cè)細(xì)胞凋亡。結(jié)果成功構(gòu)建p CDH-Flag-SV40LT-GFP慢病毒表達(dá)載體,獲得滴度為4.03×108pfu/ml的病毒。分離E9.5d MEF細(xì)胞并感染上述病毒,陽性細(xì)胞擴(kuò)大培養(yǎng)并穩(wěn)定傳代50代以上,成功建立了永生化的MEF細(xì)胞系。GPS2敲除MEF細(xì)胞的增殖能力明顯低于野生型,而二者由于血清撤除所引起的凋亡并無明顯差異。結(jié)論敲除GPS2抑制MEF細(xì)胞的增殖。
[Abstract]:Objective to establish immortalized GPS2 wild and knockout mouse embryonic fibroblast cell lines with lentivirus overexpression of SV40 large T antigen (SV40LTT), and to detect the effect of GPS2 knockout on MEF proliferation and apoptosis. Methods the expression vector of p CDH-Flag-SV40LT-GFP lentivirus was constructed. MEF cells were isolated and infected with SV40LT lentivirus and cultured for more than 50 generations. The GFP positive cells, which are still alive and in good condition, are regarded as immortalized successful MEF cells. The proliferation of immortalized MEF cells was detected by high content cell imaging analyzer. Annexin 鈪，

本文編號(hào)：1494115