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

发布时间：2018-06-18 12:17

本文选题：神经干细胞 + 慢病毒感染　；参考：《中国组织工程研究》2017年21期

【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。
[Abstract]:Background: neural stem cells (NSCs), as a hot topic in neuroscience research in recent years, have a broad application prospect in the treatment of nervous system injury. However, how to obtain a large number of purified and homogeneous terminal nerve cells is a difficult point in this field. The use of intracellular fluorescence reporting system to trace the differentiation of neural stem cells and obtain a single type of terminal nerve cells provided a feasible solution to this difficulty. Aim: to investigate the value of fluorescent reporting system driven by promoter of astrocyte-specific marker GFAP in neural stem cell differentiation. Methods: the brain cortex of mouse embryos was isolated and cultured by mechanical digestion and blow up. The expression of nestin was detected by immunofluorescence staining to determine the neural stem cells. Mouse neural stem cells were infected with lentivirus carrying pL V / Final-neo-GFAPN promoter -dT omato vector. The purified neural stem cells were obtained after G418 screening for 14 days, and then induced to differentiate into astrocytes. The changes of red fluorescent T omatoin were observed by microscope. On the 13th day of induction, GFAP antibody was restained to the cells expressing red fluorescence by cell immunofluorescence technique. Results and conclusion the primary isolated mouse neural stem cells were nestin positive and infected with lentivirus for 14 days. After screening for 14 days, purified neural stem cells with G418 resistance were obtained. A large amount of red fluorescence was observed under microscope, and there was a high consistency between GFAP restaining and red fluorescence. The mouse neural stem cells expressing neo-GFAPP promoter -dT omato vector were successfully obtained in vitro. This cell can trace the specific expression of GFAP gene in vitro, which provides a powerful tool for the study of neural stem cell differentiation mechanism, cell transplantation and tissue engineering product development.
【作者单位】：中山大学附属中山医院肿瘤研究所;中山大学附属中山医院分子诊断中心;中山大学干细胞与组织工程研究中心;
【基金】：广东省自然科学基金(2014A030310013) 中国博士后基金(2014M562244)~~
【分类号】：R318

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