【摘要】 目的利用不同的表达载体(阳离子脂质体及腺相关病毒rAAV-2-EGFP)转染神经干细胞,比较转染的效率,长期稳定性以及对神经干细胞活性的影响;再用不同转染方法(贴壁法及悬浮转染法)转染神经干细胞,比较转染的效率;为神经干细胞的标记以及进一步移植示踪寻找一种更为理想的方法。方法通过体外分离,培养孕12d胚鼠脑中神经干细胞,分别采用腺相关病毒与阳离子脂质体转染神经干细胞,于转染后36h,1周、2周、1月、6月采用荧光显微镜下计数绿色荧光细胞数目及比例,稳定表达后利用流式细胞仪检测稳定转染的效率,MTT法测定2种方法对神经干细胞增殖活性的影响,利用腺相关病毒转染对比贴壁法与悬浮法的转染效率。结果腺相关病毒转染后36h开始出现荧光,1周后转染效率为61.17%,明显高于脂质体转染的38.70%,并能长期稳定表达(6月),对神经干细胞的生长及增殖无明显的影响;悬浮法和贴壁法转染的效率分别为(60.1±3.5)%、(27.4±4.2)%,2组比较有显著性差异(P<0.05)。结论利用腺相关病毒及采用悬浮法转染神经干细胞是一种比较好的转染标记方法,适合干细胞移植后长期示踪检测。更多还原

【Abstract】 Objective To compare the effects of different transfection methonds and green fluorescent protein(GFP)expression vectors on transfection efficiency in mouse neural stem cells, and offer a tool for transplanted cell tracking in vivo.Methods After NSCs were isolated and cultivated from embryo mice which gestational age was 12 weeks, the NSCs were transfected respectively by rAAV-2-eGFP and liposome2000. At the different time point after transfection (24 h, 36 h, 48 h, 7 d, 14 d 30 d, 180 d), we counted the proportion of transfection under the fluorescence microscope, detecting the transfection efficiency with flow cytometry and the cytoactive with MTT to compare the two mothods. And the transfection efficiency was compared between suspension and adherent transfection.Results The NSCs began to appear green fluorescence after 36 h transfected by rAAV-2-eGFP, and the proportion reached the peak at 7 d, which was 61.17%, much higher than the liposomes’, which was only 38.7%. And the green fluorescence lasted for six months with no weakness. The MTT results also showed that the transfection by rAAV-2-eGFP had no obvious effect on the cytoactive. Transfection efficiency by suspension method and adherent method was(60.1±3.5)% and (27.4±4.2)%, and separately with the difference being statistically significant (P<0.05).Conclusions Transfection of mouse neural stem cells with suspension method and rAAV-2-eGFP could obtain higher transfection efficiency in NSCs, which is fit for the long-term labeling and detection of the NSCs after transplantation.更多还原