神经干细胞神经分化示踪中GFAP启动子驱动荧光报告系统的价值  

The value of GFAP promoter driven fluorescent reporter system in the neural differentiation tracing of neural stem cells

作　　者：陈京[1,2] 余伟华[3] 李付贵[1] 

机构地区：[1]中山大学附属中山医院肿瘤研究所,广东省广州市510080 [2]中山大学附属中山医院分子诊断中心,广东省中山市528403 [3]中山大学干细胞与组织工程研究中心,广东省广州市510080

出　　处：《中国组织工程研究》2017年第21期3370-3375,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金(2014A030310013);中国博士后基金(2014M562244)~~

摘　　要：背景:神经干细胞作为近年来神经科学研究的热点,在神经系统损伤治疗方面具有广阔的应用前景,但如何获取大量纯化且特征均一的终末神经细胞是这一领域的难点。利用细胞内荧光报告系统示踪神经干细胞分化的过程,并获得纯化的单一类型终末神经细胞为这一难点的解决提供了可行的方案。目的:探讨携带星形胶质细胞特异标志物GFAP基因的启动子驱动的荧光报告系统在神经干细胞神经分化示踪中的价值。方法:原代分离小鼠胚胎的脑部皮质,经机械消化和吹打后悬浮培养,免疫荧光染色检测其特异标志物Nestin的表达以确定神经干细胞。再将携带pL V/Final-neo-GFAP(promoter)-dT omato载体的慢病毒感染小鼠神经干细胞,遗传霉素G418筛选14 d后获得纯化神经干细胞,然后诱导其向星形胶质细胞分化,显微镜观察细胞红色荧光(dT omato)的变化。诱导第13天采用细胞免疫荧光技术对表达红色荧光的细胞行GFAP抗体复染。结果与结论:(1)原代分离获得的小鼠神经干细胞呈Nestin表达阳性;(2)慢病毒感染并筛选14 d后得到具有G418抗性的纯化神经干细胞;(3)该细胞经神经诱导分化后,显微镜下观察到红色荧光大量表达,且GFAP复染与红色荧光具有非常高的一致性;(4)实验成功地获得了体外表达neo-GFAP(promoter)-dT omato载体的小鼠神经干细胞,该细胞可以体外示踪GFAP基因的特异表达,为神经干细胞的定向神经分化机制、细胞移植及组织工程产品开发等研究提供了有力的工具。BACKGROUND： Neural stem cells, as a hot topic in neuroscience research, have a wide application prospect in the treatment of neurological damage, but how to obtain a large number of terminally differentiated and purified nerve cells with homogeneous features is a difficult problem in this field. The use of intracellular fluorescence reporter system to track the process of neural stem cell differentiation and obtain a single kind of terminally differentiated and purified nerve cells provides a viable option. OBJECTIVE： To explore the value of GFAP promoter-driven fluorescence reporter system in tracing the neural differentiation of neural stem cells（NSCs）. METHODS： Cerebral cortex of mouse embryos were primarily dissociated and sent for digesting and pipetting mechanically before suspension culture, followed by immunofluorescence staining of Nestin to identify their biological characteristics. Lentivirus carrying p LV/Final-neo-GFAP（promoter）-d Tomato vector was employed to infect above-mentioned NSCs, and Geneticin（G418） was used to obtain purified NSCs at 14 days. Subsequently the purified cells were induced to differentiate into astrocyte-like cells; meanwhile red fluorescence changes in cells were observed by microscopy. The red fluorescent cells were then subjected to perform immunofluorescence staining at 13 days after induction. RESULTS AND CONCLUSION： The expression of Nestin in the isolated primary cells was strongly positive. Purified NSCs were obtained by lentivirus infection and subsequent G418 resistance selection at 14 days. After induced into astrocyte-like cells, the red fluorescence was observed in the cells under the microscope and furthermore, GFAP staining was also positive. Mouse NSCs carrying neo-GFAP（promoter）-d Tomato were successfully obtained. The cells could express d Tomato under the control of GFAP promoter, which provides a powerful tool for research on NSC differentiation mechanism, neural transplantation and tissue engineering product development.

关 键 词：神经干细胞 慢病毒感染 神经胶质原纤维酸性蛋白质 组织工程 干细胞 分化 慢病毒 红色荧光蛋白 胶质纤维酸性蛋白 广东省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]