神经生长因子重组反转录病毒载体在神经干细胞中表达.docVIP

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 何峻峰1，顾国妹1，龙大宏2 1上海市浦东医院，上海市? 2013002广州医科大学人体解剖教研室，广东省广州市? 510182 Expression of recombinant retroviral vector carrying nerve growth factor gene in neural stem cells He Jun-feng1, Gu Guo-mei1, Long Da-hong2 1Pudong Hospital, Shanghai 201300, China2Department of Anatomy, Guangzhou Medical University, Guangzhou 510182, Guangdong Province, China 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。 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程 全文链接： 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； ?? Abstract： 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 cell genome, which is suitable for gene therapy.OBJECTIVE:?To study the gene expression of recombinant retroviral vector carrying rat NGF gene in neural stem cells.METHODS:?The rat NGF gene was inserted into a retroviral vector pLEGFP-N1, which was transferred into packaging cells PT67 by Lipofectamine 2000. The positive clones in virus supernatant were collected by G418 selection and used to infect neural stem cells. After that, the NGF expression was tested by enzyme linked immunosorbent assay and the biological competence by PC12 cells, and then morphological change of neural stem cells and cell typing were examined by fluorescent microscope.RESULTS AND CONCLUSION:?The neural stem cells could express extrinsic source NGF protein after the recombinant plasmid