小鼠水通道蛋白1基因慢病毒载体构建及其在神经膜细胞中的表达  

Construction of lentiviral vector containing mouse aquaporin-1 gene and its expression in Schwann cells

作　　者：章杰[1,2] 江华[1] 汪汇[1] 方帆[1] 宋艳玲[1] 芦立轩[1] 

机构地区：[1]第二军医大学长征医院整形外科,上海200003 [2]南昌大学第一附属医院整形美容科,南昌330006

出　　处：《第二军医大学学报》2013年第2期199-204,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(31271264);上海市科委基础研究重点课题(08JC1407100)~~

摘　　要：目的构建表达小鼠水通道蛋白1(AQP1)基因的慢病毒载体,体外感染原代培养的C57BL/6小鼠神经膜细胞,观察是否提高AQP1的表达,为进一步研究AQP1与周围神经系统损伤后水肿的关系奠定基础。方法将小鼠AQP1基因克隆到慢病毒pCDH-CMV-MCS-EF1-copGFP载体,通过PCR和测序鉴定获得连接正确的克隆。将鉴定后的重组表达质粒pCDH-CMV-MCS-EF1-copGFP-AQP1与包装质粒psPAX2、pMD共转染293T细胞,制备携带AQP1基因的慢病毒lentivirus-AQP1。体外培养C57BL/6小鼠的神经膜细胞,将lentivirus-AQP1感染神经膜细胞,RT-PCR和蛋白质印迹法检测感染后神经膜细胞AQP1 mRNA和蛋白的表达情况。结果构建的慢病毒载体pCDH-CMV-MCS-EF1-copGFP-AQP1经PCR鉴定和测序正确。慢病毒lentivirus-AQP1感染神经膜细胞后AQP1表达增加(P<0.05)。结论成功构建了小鼠AQP1基因的慢病毒表达载体,该载体能有效感染神经膜细胞,使AQP1 mRNA和蛋白表达水平增高。Objective To construct a lentiviral vector carrying mouse aquaporin-1 （AQP1） gene and use it for infecting Sehwann cells of C57BL/6 mouse, so as to provide AQP1＋ Schwann cells for further studying the relationship of AQP1 with peripheral nerve system injury edema. Methods Mouse AQP1 gene was inserted into lentiviral vector pCDH-CMV-MCS-EF1- copGFP, and the products were confirmed by PCR and sequencing analysis, pCDH-CMV-MCS-EFI-copGFP-AQP1 and the virus packaging plasmids psPAX2 and pMD were cotransducted into 293T cells to prepare lentivirus-AQP1, and the latter was used to infect C57BL/6 mouse Schwann cells in vitro. The expression of AQP1 mRNA and protein was detected by quantitative real-time PCR and Western blotting analysis in infected mouse Schwann ceils. Results PCR and sequencing revealed that the pCDH-CMV-MCS-EFI-copGFP-AQP1 plasmids were successfully constructed. The expression of AQP1 mRNA and protein in Schwann cells was significantly increased than that in cells infected with control lentiviruses （P〈0.05）. Conclusion We have successfully constructed a recombinant lentivirus carrying AQP1 gene, which can be used to infect mouse Schwann cells, leading to increase of AQP1 mRNA and protein expression.

关 键 词：水通道蛋白1 过表达 慢病毒 神经膜细胞 

分 类 号：R349.83[医药卫生—基础医学]