| 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。 |
| 关键词: 水通道蛋白1 过表达 慢病毒 神经膜细胞 |
| DOI：10.3724/SP.J.1008.2013.00199 |
| 投稿时间：2012-09-06修订日期：2012-10-11 |
| 基金项目:国家自然科学基金(31271264),上海市科委基础研究重点课题(08JC1407100). |
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| Construction of lentiviral vector containing mouse aquaporin-1 gene and its expression in Schwann cells |
| ZHANG Jie1,2,JIANG Hua1*,WANG Hui1,FANG Fan1,SONG Yan-ling1,LU Li-xuan1 |
(1. Department of Plastic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
2. Department of Plastic Surgery, The First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
*Corresponding author.) |
| Abstract: |
| Objective To construct a lentiviral vector carrying mouse aquaporin-1 (AQP1) gene and use it for infecting Schwann 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-EF1-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 cells. Results PCR and sequencing revealed that the pCDH-CMV-MCS-EF1-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. |
| Key words: aquaporin 1 over-expression lentivirus Schwann cells |
