绿色荧光蛋白作为神经干细胞示踪标记的实验研究

目的研究携带绿色荧光蛋白(green fluorescent protein,GFP)的腺病毒感染神经干细胞(neural stem cells,NSCs)后的荧光稳定性和持久性及其对NSCs生物学特性的影响;探讨利用其作为NSCs示踪标记的可行性.方法利用GFP-腺病毒空载体感染NSCs(NSCs-GFP),经G418筛选并连续传代,在相差显微镜下观察未感染NSCs和感染NSCs的形态以及在荧光显微镜下观察感染NSCs及其诱导分化后的GFP表达情况;并检测NSCs-GFP的细胞活性和细胞周期.调整GFP标记神经干细胞浓度为1×105/μl,将其植入大鼠侧脑室内,观察其在脑内的表达.结果未感染NSCs和感染NSCs的形态学、细胞活性和细胞周期等无明显差异(P＞0.05).GFP在诱导分化后的NSCs-GFP子代细胞中有高表达,并且第1代和第20代NSCs-GFP的绿色荧光无明显差别.在植入侧侧脑室周围可见绿色荧光的强表达,且持续时间较长.结论 GFP荧光稳定持久,对转染细胞的生物学特征无明显影响,并且移植后的荧光呈长时间强表达,因此可以将其作为神经干细胞的示踪标记,这将有利于神经干细胞移植后的可塑性研究.

Green fluorescent protein as a marker of neural stem cells

Objective To study the fluorescent stabilization and persistence of green fluorescent protein (GFP) and its effect on bionomics of the infected neural stem cells (NSCs), and explore the feasibility of NSCs marked by GFP (NSCs-GFP). Methods NSCs were infected by GFP-adenovirus carrier. After the infected cells were selected by G418, GFP expression of NSCs-GFP and differentiated NSCs-GFP were detected under phase contrast microscope and fluorescent microscope. The morphological feature, the cytoactive, and cell cycle of the uninfected cells were compared with those of the infected cells. NSCs-GFP was transplanted into the ventricle of rats and the expression of GFP in the brain was examined under fluorescent microscope. Results The morphological feature of the uninfected NSCs was similar to that of the NSCs-GFP, and there were no significant differences in the cytoactive and cell cycle between the NSCs and the NSCs-GFP (P>0.05). The expression of GFP was located around the ventricle and persisted for a long time. Conclusion The fluorescence of GFP is stable and persistent and the GFP has no negative effects on the NSCs-GFP. It is an ideal maker of neural stem cells and could be used for the study of NSCs transplantation.