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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1.0×107~1.0×108U/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。
Objective To investigate the effect of CRMP4 on neurite outgrowth in hippocampus. Methods The target gene fragment of CRMP4 was amplified by PCR and ligated with the viral vector. After being transfected into 293T cells by PCR, digestion and identification, the lentiviral vector was prepared and then infected into hippocampal neurons to observe the changes of neurite outgrowth , And quantitative analysis. Results The target gene of CRMP4 was amplified and the CRMP4 lentiviral vector was successfully constructed. The titer of the virus was 1.0 × 107-1.0 × 108U / ml. The control cells transfected with empty plasmid showed GFP expression in the somatic cells and neurites, With the development of polarization, differentiation of dendrites and axons, protuberances continue to extend; over-expression of CRMP4, we can see proton neurite extension, branching than the control group increased; quantitative results show that the total length of neuronal processes including dendrites and Axons were increased compared with the control group, the number of branches also increased compared with the control group, the difference was significant (P <0.05). Conclusion The lentiviral vector carrying rat CRMP4 gene was constructed successfully. Overexpression of CRMP4 gene can promote neurite outgrowth.