慢病毒介导人神经营养素3基因在许旺细胞中的表达  

Expression of lentivirus-mediated neurotrophin-3 gene in Schwann cells

作　　者：袁健东[1] 傅强[1] 连小峰[1] 侯铁胜[1] 赵杰[1] 

机构地区：[1]上海长海医院骨科,上海市200433

出　　处：《中国组织工程研究与临床康复》2009年第32期6327-6331,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目"组织工程化自体雪旺氏细胞移植治疗脊髓损伤的实验研究(30571887)";上海市青年科技启明星计划资助项目"组织工程化基因修饰雪旺氏细胞移植治疗脊髓损伤的实验研究(06QA14068)"~~

摘　　要：背景:神经营养素3是目前发现的在脊髓损伤修复中作用最强的神经营养因子,它能有效促进再生轴突穿越胶质瘢痕组织,从而修复脊髓损伤。目的:构建含有人神经营养素3基因的重组慢病毒载体(LV-hNT3),观察转染后人神经营养素3基因在许旺细胞中的表达。设计、时间及地点:观察性实验,于2007-06/2008-03在长海医院中心实验室完成。材料:取新生3d的SD乳鼠双侧坐骨神经,用于许旺细胞的培养及鉴定;慢病毒三质粒系统pGC-E1-EGFP,pHelper1.0和pHelper2.0为上海吉凯基因化学技术有限公司产品。方法:通过双限制性内切酶消化和连接的方法构建pGC-E1-hNT3-EGFP质粒,接着该质粒转化感受态的大肠杆菌E.coliDH5α,通过PCR及基因测序鉴定阳性克隆,再经Lipofectamine2000将pGC-E1-hNT3-EGFP,pHelper1.0和pHelper2.0三质粒系统共转染293T细胞包装病毒,按照病毒感染复数=1,4,8,10,12加入预先混好重组病毒完全培养液2mL,通过增强型绿色荧光蛋白的表达测定收集的病毒滴度。将慢病毒转染许旺细胞,以未经转染的许旺细胞和经空载的慢病毒转染的许旺细胞为对照组。主要观察指标:慢病毒转染许旺细胞后,检测转染效率,通过实时荧光聚合酶链反应和蛋白免疫印迹检测人神经营养素3在许旺细胞中的表达。结果:实验中重组质粒的外源基因序列与GeneBank中的人神经营养素3阅读框架序列完全一致;浓缩后病毒滴度为5×107TU/L,LV-hNT3感染许旺细胞后,许旺细胞发出明亮的绿色荧光,当感染复数=10时转染效率最高达85%,实时荧光PCR检测表明人神经营养素3mRNA在人神经营养素3-许旺细胞中高效表达,而对照组中未表达,蛋白免疫印迹证实人神经营养素3在许旺细胞中表达。结论:实验构建的含有人神经营养素3基因的慢病毒载体能感染许旺细胞并且高效表达人神经营养素3。BACKGROUND： Neurotrophin-3 is found in the repair of spinal cord injury in the role of the strongest neurotrophic factor. It can effectively promote axonal regeneration through the glial scar tissue in repairing spinal cord injury. OBJECTIVE： To construct recombinant lentiviral vectors for gene delivery of homo sapiens neurotrophin-3 （hNT3）, and to investigate the expression of hNT3 gene in Schwann cells after transfection. DESIGN, TIME AND SETTING： Observational experiment was performed from June 2007 to March 2008 at the Central Laboratory of Changhai Hospital. MATERIALS： Bilateral sciatic nerves were harvested from 3-day-old Sprague Dawley rats for culture and identification of Schwann cells. Three-plasmid lentivirus systems： pGC-E1-EGFP, pHelper 1.0 and pHelper 2.0 were gained from Shanghai Chemical Technology Co., Ltd. METHODS： pGC-EI-hNT3-EGFP plasmid was constructed by double restriction enzyme digestion and ligation, and then the plasmid was transformed into E.coli DH5α. Purified pGC-E1-hNT3-EGFP plasmids from the positive clones was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGC-E1-hNT3-EGFP, pHelper 1.0 and pHelper 2.0 by Lipofectamine 2000 to produce lentivirus. 2mL recombinant virus complete culture solution was added according to multiplicity of infection=1, 4, 8, 10, 12. The titer of virus was tested according to the expression level of enhanced green fluorescent protein. The control groups were Schwann cells and Schwann cells transfected by no-loaded lentivirus. MAIN OUTCOME MEASURES： The lentiviruses were transduced to Schwann cells, and the transfection efficiency was examined by flow cytometry, the overexpression of hNT3 was determined by Real-time PCR and Western blotting. RESULTS： The exogenous gene sequence of the recombinant hNT3 was completely in accordance with that of its open reading frame in GeneBank. The titer of concentrated virus was 5×10^7 TU/L. After recombinant LV-hNT3 infection, Schwann cells gave off strikingly bri

关 键 词：慢病毒 许旺细胞 基因转染 神经营养素3 

分 类 号：R394.2[医药卫生—医学遗传学]