Rho激酶调节大鼠海马神经元突起生长和微管重排的研究

发布时间：2018-05-08 19:37

本文选题：Rho激酶 + 突起　；参考：《暨南大学》2007年硕士论文

【摘要】：目的：探讨Rho激酶通路对神经元突起生长和微管重排的调节。材料和方法：以大鼠海马神经元为对象，用Rho激酶促进剂LPA和Rho激酶抑制剂Y27632干预，原子力显微镜观察细胞突起的生长，并提取突起进行定量分析，共聚焦激光扫描显微镜观察微管的重排。结果：(1)neurobasal无血清培养基培养获得的神经元纯度较高，且活力旺盛，贴壁后6-11d的细胞适宜实验观察。(2)原子力显微镜的观察：LPA处理组神经元突起的数量减少，1-3级突起的数目均减少明显(P＜0.05)；LPA预处理后再用Y27632进行干预，一级突起的数目明显增多，其中二级突起增多明显(P＜0.05)；提取突起的光密度测定显示，LPA处理后的细胞明显降低(P＜0.05)，LPA预处理后用Y27632干预组的细胞较LPA组明显升高(P＜0.05)，而且超过对照组水平，但无显著差异。(3)共聚焦激光扫描显微镜观察：神经元胞体、突起和生长锥均有清晰的微管。LPA处理组的神经元胞体内无清晰可见呈鸟巢样外观的丝网状微管结构，取而代之的是不规则的微管样结构；突起较短，而且突起远端的荧光较淡，突起内微管的排列出现发辫样改变；生长锥失去原有的典型的生长锥样结构，仅见突起细小的末端，其中的微管大部分消失。LPA预处理后用Y27632干预组的神经元胞体内可见较清晰的丝网状微管结构，微管深入细胞的突起；突起内的微管仍可见较大的空隙，但明显较LPA组排列规整；突起末端生长锥基本保持了原有的形态，其中可见较清晰的微管，，它们直行深入生长锥或者弯曲走行。结论：(1)从新生大鼠大脑可成功分离获得大量海马神经元，在neurobasal无血清培养基中生长良好。(2)激活Rho激酶通路可诱导神经元突起坍塌，表现在各级突起数目的减少和长度的缩短，抑制Rho激酶通路可促使神经元突起各级分支数目的增加以及突起的生长。(3)Rho激酶不但通过调节微管重排参与突起的生长延长，还参与突起的分支形成，抑制Rho激酶的活性基本能逆转LPA对突起生长的坍塌作用，但并不能完全逆转LPA诱导的微管重排。
[Abstract]:Objective: to investigate the regulation of Rho kinase pathway on neurite growth and microtubule rearrangement. Materials and methods: rat hippocampal neurons were treated with Rho kinase enhancer LPA and Rho kinase inhibitor Y27632. The neurites were observed by atomic force microscope and the processes were extracted for quantitative analysis. The rearrangement of microtubules was observed by confocal laser scanning microscope. Results the neurons cultured in serum-free medium were of high purity and high activity. The number of neuronal processes in the treatment group decreased significantly (P < 0.05) after pretreatment with Y27632, and the number of first-order protrusions increased significantly, and the number of neuronal processes decreased significantly after pretreatment with Y27632 (P < 0.05), and the number of neuronal processes decreased significantly after treatment with LPA (P < 0.05), and Y27632 was used in the treatment group. The number of secondary processes increased significantly (P < 0.05), and the optical density of the extracted processes showed that the cells treated with LPA-treated with Y27632 were significantly lower than those in the LPA group (P < 0.05), and the level of cells in the treatment group was higher than that in the control group, and the number of cells in the treatment group was significantly higher than that in the control group, and that in the treatment group with Y27632 was significantly higher than that in the LPA group. But there was no significant difference. (3) Confocal laser scanning microscopy showed that there were no clear filamentous microtubules in the neuronal bodies, processes and growth cones in the LPA treated group, and there were no clear filamentous microtubules in the neuronal bodies with the appearance of bird's nest. Instead of the irregular tubule-like structure, the protrusions are shorter, and the fluorescence at the distal end of the protuberances is lighter, and the arrangement of the microtubules in the protuberances changes like braided; the growth cones lose their original typical growth cone-like structures and only see the small ends of the protuberances. Most of the microtubules disappeared. After pretreatment with Y27632, clear filamentous microtubule structure was found in the neurons of Y27632 intervention group, and the microtubules in the process could still be seen larger voids, but the arrangement of the microtubules was more regular than that in the LPA group. The shape of the growth cone at the end of the protuberance is basically the same, and the clear microtubules can be seen, which go directly into the growth cone or bend. Conclusion: 1) A large number of hippocampal neurons were successfully isolated from the brain of newborn rats. The neurons grew well in neurobasal serum-free medium. The activation of Rho kinase pathway could induce neuronal neurites to collapse, which was manifested by the reduction of the number of processes and the shortening of length. Inhibition of Rho kinase pathway can promote the increase of the number of branches of neuronal processes and the growth of protrusions. Rho kinases not only participate in the extension of processes by regulating microtubule rearrangement, but also participate in the formation of branches of processes. Inhibition of the activity of Rho kinase could reverse the collapse of LPA on the growth of process, but it could not completely reverse the microtubule rearrangement induced by LPA.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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