神经生长因子重组反转录病毒载体在神经干细胞中的表达  

Expression of recombinant retroviral vector carrying nerve growth factor gene in neural stem cells

作　　者：何峻峰 顾国妹 龙大宏[2] 

机构地区：[1]上海市浦东医院,上海市201300 [2]广州医科大学人体解剖教研室,广东省广州市510182

出　　处：《中国组织工程研究》2014年第19期3100-3104,共5页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金(04009566);广东省医学科研基金(A2003276);广州市科技计划项目(041011)~~

摘　　要：背景:神经生长因子属于生物大分子,难以透过血脑屏障,而反转录病毒载体能稳定地将外源基因插入并整合到宿主细胞基因组内,适于作为基因治疗的载体。目的:探讨神经生长因子基因重组反转录病毒表达载体在神经干细胞中的表达情况。方法:将SD大鼠神经生长因子基因重组反转录病毒表达载体pLEGFP-NGF通过脂质体Lipofectamine 2000转染包装细胞PT67,经G418筛选后,收集阳性克隆病毒上清,用于感染神经干细胞,用ELISA检测神经生长因子基因的表达,并利用PC12细胞检验神经生长因子的生物学活性,同时观察神经生长因子对神经干细胞存活、分化的影响。结果与结论:重组了神经生长因子基因的反转录病毒液感染神经干细胞后能表达外源性神经生长因子蛋白,该蛋白能促使PC12细胞数量增多,突起明显变长,并能增加神经干细胞的存活数量,促进神经干细胞分化。结果说明整合了神经生长因子基因的神经干细胞能表达外源性神经生长因子,表达的神经生长因子对神经干细胞的存活及分化均有促进作用。BACKGROUND：Nerve growth factor （NGF） belongs to a biological macromolecule that is difficult to pass through the blood-brain barrier. However, a retroviral vector carrying exogenous gene can be stably inserted and integrated into the host cellgenome, which is suitable for gene therapy. OBJECTIVE：To study the gene expression of recombinant retroviral vector carrying rat NGF gene in neural stem cels. METHODS： The rat NGF gene was inserted into a retroviral vector pLEGFP-N1, which was transferred into packaging cels PT67 by Lipofectamine 2000. The positive clones in virus supernatant were colected by G418 selection and used to infect neural stem cels. After that, the NGF expression was tested by enzyme linked immunosorbent assay and the biological competence by PC12 cels, and then morphological change of neural stem cels and celltyping were examined by fluorescent microscope. RESULTS AND CONCLUSION：The neural stem cels could express extrinsic source NGF protein after the recombinant plasmid was infected into neural stem cels. The PC12 cels increased in the experimental group and stretched out long neurites. And the NGF protein could maintain the neural stem cellsurvival and stimulate their differentiation. These findings suggest that the neural stem cels carrying extrinsic source NGF gene could express NGF successfuly, and the NGF protein induced the survival and differentiation of neural stem cels.

关 键 词：干细胞 神经干细胞 神经生长因子 反转录病毒载体 PC12细胞 基因重组 大鼠 广东省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]