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坍塌反应调节蛋白5促进海马神经元突起生长
Collapsin response mediator protein 5 accelerates neurite outgrowth in hippocampal neurons
目的 探讨坍塌反应调节蛋白5(CRMP5)对神经元突起生长的作用。方法 构建CRMP5真核表达载体，采用基因转染、实时定量PCR和免疫印迹技术评估CRMP5基因表达；以空载体为对照组，设3个复孔，用时差成像技术和突起提取技术观察和检测原代培养海马神经元突起的生长。结果 成功构建携带增强型绿色荧光蛋白（EGFP）标签蛋白的CRMP5的真核表达载体。脂质体转染技术可成功把CRMP5基因导入细胞，转染的细胞CRMP5表达高于空载体对照组；CRMP5蛋白表达于神经元的胞体和突起，尤其是胞体、突起起始处和突起末端高表达。过表达CRMP5可明显促进突起生长，主要表现为突起的生长，并形成丰富的侧枝；定量结果显示，CRMP5过表达的细胞突起的长度逐渐延长，而且较空载体转染细胞增多，差异显著(P<0.01)。导入CRMP5的细胞突起提取液的吸光度较对照细胞明显升高(P.<0.01)。 结论 CRMP5能促进神经元突起及其分支的生长。
Objective To investigate function of collapsin response mediator protein 5(CRMP5) on neurite outgrowth. Methods The CRMP5 eukaryotic expression vector was constructed and transfected into hippocampal neurons. The gene transfection, Real-time quantitative PCR and Western blotting were used to detect expression of CRMP5 protein. The lapse-time imaging and neurite extraction were utilized to show neurite outgrowth and differentiation and 3 double-pored were performed, compared with the vector without CRMP5 gene. Results It was successful to construct the CRMP5 eukaryotic expression vector with an EGFP tag. The lipofectamine effectually transfected CRMP5 into cultured neurons, and the CRMP5 protein was expressed successfully more than the control cells. CRMP5 protein was abundant in cell body, initiation and end of neurites. Overexpression of CRMP5 in neuronal cells significantly promoted outgrowth neurites, and led to the formation of longer neurites with more branches. Accompanying rapid outgrowth of neurites, branches from original neurites were contributed to form a network. The results of neurite length and extraction showed that neurons overexpressing CRMP5 were possessed more and longer neurites (P<0.01), compared with control cells. Conclusion The results suggest that CRMP5 accelerates not only axonal growth but also branching.
坍塌反应调节蛋白5 / 神经元 / 突起 / 生长 / 基因转染 / 真核表达 / 免疫印迹法 / 大鼠
Collapsin response mediator protein 5 / Neuron / Neurite / Outgrowth / Gene transfection / Eukaryotic expression / Western blotting / Rat
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