CRMP4慢病毒载体的构建及其对神经元突起生长的影响

张丹丹; 谭明会; 叶永恒; 陈克恩; 李素梅; 武凤鸣; 郭国庆

doi:10.13418/j.issn.1001-165x.2015.03.016

中国临床解剖学杂志 ›› 2015, Vol. 33 ›› Issue (3) : 305-310. DOI: 10.13418/j.issn.1001-165x.2015.03.016

实验研究

CRMP4慢病毒载体的构建及其对神经元突起生长的影响

张丹丹¹，　谭明会^1,2，　叶永恒¹，　陈克恩^1,3，　李素梅¹，　武凤鸣¹，　郭国庆^1,2,4

作者信息 +

Construction of CRMP4 lentiviral vector and its influence on neurite development

ZHANG Dan-dan^1,2, TAN Ming-hui^1,2, YE Yong-heng ¹, CHEN Ke-en^1,3, LI Su-mei¹, WU Feng-ming¹, GUO Guo-qing^1,2,4

Author information +

文章历史 +

摘要

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。

Abstract

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.

导出引用

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 https://doi.org/10.13418/j.issn.1001-165x.2015.03.016

Construction of CRMP4 lentiviral vector and its influence on neurite development[J]. Chinese Journal of Clinical Anatomy. 2015, 33(3): 305-310 https://doi.org/10.13418/j.issn.1001-165x.2015.03.016

参考文献

[1] Barzilai A, Zilkha-Falb R, Daily D, et al. The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity [J]. Neural Transm Suppl, 2000, (60):59-76.
[2] Hall C, Brown M, Jacobs T, et al. Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase [J]. Biol Chem, 2001, 276(46):43482-43486.
[3] Brot S, Rogemond V, Perrot V, et al. CRMP5 interacts with tubulin to inhibit neurite outgrowth, thereby modulating the function of CRMP2 [J]. Neurosci, 2010, 30(32): 10639-10654.
[4] Fukata Y, Itoh TJ, Kimura T, et al. CRMP-2 binds to tubulin heterodimers to promote microtubule assembly [J]. Nat Cell Biol, 2002, 4(8): 583-591.
[5] Katzourakis A, Tristem M, Pybus OG, et al. Discovery and analysis of the first endogenous lentivirus [J]. Proc Natl Acad Sci U S A, 2007, 104(15): 6261-6265.
[6] Cockrell AS, Kafri T. Gene delivery by lentivirus vectors [J]. Mol Biotechnol, 2007, 36(3): 184-204.
[7] Albright TD, Jessell TM, Kandel ER, et al. Neural science: a century of progress and the mysteries that remain [J]. Cell, 2000, 100 Suppl: S1-55.
[8] Lowery LA, Van Vactor D. The trip of the tip: understanding the growth cone machinery [J]. Nat Rev Mol Cell Biol, 2009, 10(5): 332-343.
[9] Geraldo S, Gordon-Weeks PR. Cytoskeletal dynamics in growth-cone steering [J]. Cell Sci, 2009, 122(Pt 20): 3595-3604.
[10]Yoshimura T, Kawano Y, Arimura N, et al. GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity [J]. Cell, 2005, 120(1): 137-149.
[11] Rosslenbroich V, Dai L, Baader SL, et al. Collapsin response mediator protein-4 regulates F-actin bundling [J]. Exp Cell Res, 2005, 310(2): 434-444.
[12]Liu BP, Strittmatter SM. Semaphorin-mediated axonal guidance via Rho-related G proteins[J]. Curr Opin Cell Biol, 2001, 13(5): 619-626.
[13] Arimura N, Kaibuch K. Neuronal polarity: from extracellular signals to intracellular mechanisms[J]. Nat Rev Neurosci, 2007, 8(3): 194-205.
[14] Kowara R, Menard M, Brown L, et al. Co-localization and interaction of DPYSL3 and GAP43 in primary cortical neurons[J]. Biochem Biophys Res Commun, 2007, 363(1): 190-193.