收稿日期: 2014-10-17
网络出版日期: 2015-07-24
基金资助
国家自然科学基金( 31170941)；广东省自然科学基金项目(S2013010014191)；中国博士后基金( 2014M562252)；广东省医学科研基金( A2014382)
Construction of CRMP4 lentiviral vector and its influence on neurite development
Received date: 2014-10-17
Online published: 2015-07-24
目的 探讨CRMP4对海马神经元突起生长的影响。 方法 利用PCR技术扩增CRMP4目的基因片段，与病毒载体连接，经过PCR、酶切和鉴定后与包装质粒共转293T细胞，制备慢病毒载体，之后感染海马神经元，观察神经元突起生长的变化，并进行定量分析。 结果 扩增出CRMP4目的基因，成功构建CRMP4慢病毒载体，病毒滴度为1.0×107~1.0×108 U/ml；对照细胞转染空质粒后见GFP表达，分布于神经元的胞体和突起，细胞随着发育不断极化，分化出树突和轴突，突起不断延长；过表达CRMP4后，可见神经元的突起延长，分支较对照组增多；定量结果显示，神经元突起总长度包括树突和轴突均较对照组增多，分支数目亦较对照组增多，差异显著（P<0.05）。 结论 成功构建了携带大鼠CRMP4基因的慢病毒表达载体；过表达CRMP4基因可以促进神经元突起的生长。
张丹丹 , 谭明会 , 叶永恒 , 陈克恩 , 李素梅 , 武凤鸣 , 郭国庆 . CRMP4慢病毒载体的构建及其对神经元突起生长的影响[J]. 中国临床解剖学杂志, 2015 , 33(3) : 305 -310 . DOI: 10.13418/j.issn.1001-165x.2015.03.016
Objective This study aimed to investigate the influence of CRMP4 on the neurite growth in the hippocampal neuron. Methods To obtain the CRMP4 gene fragments by PCR amplification technology, then connect the gene to the viral vector. After PCR, enzyme restriction digestion and sequencing verification, the plasmids was packaged by transferring 293T cells to prepare Lentivirus Vector. The CRMP4 lentivirus vector was used to transfect hippocampal neurons cultured in vitro and neurite length of neurons was analyzed. Results The sequence of CRMP4 amplified by PCR was right and the gene fragment was inserted into lentivirus vector. The concentration of the virus was at the titer of 1.0×107~1.0×108 TU/ml. In the control cells, GFP was expressed in the neurites and cell bodies. Hippocampal neurons have two types of highly polarized cell processes, axons and dendrites with the development. When infected by CRMP4 Lentivirus Vector, the neurite outgrowth was obvious, and a lot of dendrites arose from cell bodies. The results of neurite lengths showed that over-expression CRMP4, the neutrite outgrowth was increased significantly and a lot of dendrites werefound. In cells without CRMP4 expression, the neutrites were few, and the length of neutritesdiminishedsignificantly when compared with CRMP4 over-expressing cells (P<0.05). Conclusion The lentivirus vector of CRMP4 was successfully constructed and overexpression of CRMP4 is beneficial to neurite development including neurite outgrowth and formation of branches.
Key words： CRMP4; Lentivirus; Hippocampus; Neurons; Neurite
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