CRMPs与海马神经元生长锥微管和微丝相互作用的研究

发布时间：2018-05-07 07:15

本文选题：CRMPs + 生长锥　；参考：《暨南大学》2014年硕士论文

【摘要】：目的：探讨大鼠海马神经元CRMPs与生长锥微管和微丝的相互作用。材料和方法：取孕17d的SPF级SD孕鼠，采用梯度蔗糖离心的方法提取生长锥，透射电镜和扫描电镜观察分离物的形态，免疫荧光和免疫印迹检测GAP-43的表达。采用多重免疫荧光技术分析CRMPs与微管和微丝在海马组织中以及在神经元中的共定位情况，免疫共沉淀技术确定CRMPs能否与微管和微丝发生相互作用，通过对CRMP5的干扰和过表达观察其对微管和微丝的影响。结果：（1）梯度浓度蔗糖离心把脑组织悬液分离为3层，上层密度小，浓度位于上清至0.75mol/L蔗糖之间，中层密度较大，浓度位于0.75～1.0mol/L蔗糖之间，下层密度最大，浓度位于1.0～2.66mol/L蔗糖之间；扫描电镜见分离物颗粒均为白色、大小不一、形状不规则的颗粒，上层较中、下层颗粒体积大；透射电镜见上层分离物为大量着色浅的空泡状结构，颗粒大小较均匀，中、下层分离物为大量着色深的致密颗粒及不规则形态的细胞碎片，颗粒大小不均匀；免疫荧光及免疫印迹证实分离物中均有大量GAP-43表达，上层分离物几乎无GFAP表达，中、下层有少量GFAP表达。（2）在海马组织中，CRMPs与微管和微丝的共定位均在CA1-CA3区中央部分最多，在CA1-CA3区边缘部分及CA4区较多，DG区最少，两者共定位系数差异无统计学意义；在神经元中，CRMPs与微管的共定位主要位于神经元胞质、突起主干及突起分支处，放大观察生长锥，共定位主要位于C区，P区、T区微管分布较少，CRMPs与微丝的共定位主要位于神经元胞质、突起主干、突起分支处及生长锥，放大观察生长锥，共定位位于整个生长锥，C区、T区、P区微丝分布均较多，以神经元或以生长锥为对象时，CRMPs与微丝的共定位系数均高于微管，，两者共定位系数差异均具有统计学意义；免疫共沉淀试验证实CRMPs与微管和微丝均可发生相互结合；神经元中CRMP5过表达时，微管和微丝表达量均增多，并且在P区增多最明显，CRMP5被干扰时，微管和微丝表达量均减少，也在P区减少最明显。结论：（1）梯度蔗糖离心可以依据颗粒的大小分离脑组织的不同组分，获取的分离物最上层内的组分经鉴定为生长锥；（2）CRMPs在海马组织中与微管和微丝共存相同的部位；（3）CRMPs在体内可与微管和微丝发生相互结合，并可影响微管和微丝的表达量及分布，CRMPs可能通过介导细胞骨架的相互作用来调节神经元突起的生长。
[Abstract]:Objective: To investigate the interaction of CRMPs with growth cone microtubules and microfilaments in rat hippocampal neurons. Materials and methods: The growth cone was extracted by gradient sucrose centrifugation. The morphology of the isolate was observed by transmission electron microscope (TEM) and scanning electron microscope (SEM). The expression of GAP-43 was detected by immunofluorescence and immunoblotting. The co-localization of CRMPs with microtubules and microfilaments in hippocampal tissues and neurons was analyzed by using multiple immunofluorescence techniques. The immunoprecipitation technique was used to determine whether CRMPs could interact with microtubules and microfilaments. The effects of CRMP5 on microtubules and microfilaments were observed by interference and overexpression. Results: 1) the brain suspension was separated into three layers by gradient concentration sucrose centrifugation, the upper density was small, the concentration was between the supernatant and the 0.75mol/L sucrose, the middle density was higher, the concentration was between 0.75~1.0mol/L sucrose, the lower layer density was the largest, and the concentration was between 1.0~2.66mol/L sucrose. Scanning electron microscopy (SEM) showed that the particles were all white, of different sizes and irregular in shape. The upper layer was larger than the lower, and the transmission electron microscope showed that the upper layer was of a large amount of light colored vacuole structure, the particle size was uniform, and the size of the particles was medium. In the lower layer, a large number of stained dense particles and irregular cell fragments were found, and the size of the particles was not uniform. Immunofluorescence and immunoblotting confirmed that there was a large amount of GAP-43 expression in the isolates, and there was almost no GFAP expression in the upper layer of the isolates. A small amount of GFAP was expressed in the lower layer. (2) in hippocampal tissue, the co-localization of CRMPs with microtubules and microfilaments was the most in the central part of the CA1-CA3 region, and the least in the marginal part of the CA1-CA3 area and in the CA4 area. There was no significant difference in the colocalization coefficient between the two regions. The co-localization of CRMPs and microtubules in neurons was mainly located in the cytoplasm of neurons, the main process trunk and the branches of the processes, and the growth cones were observed by magnification. The co-localization of CRMPs and microfilaments was mainly located in the cytoplasm of neurons, the main process, the branches of the processes and the growth cones, and the growth cones were observed by magnification. The co-localization coefficients of CRMPs and microfilaments were higher than those of microtubules when the neurons or growth cones were used as objects. The colocalization coefficients of CRMPs and microfilaments were higher than those of microtubules. The colocalization coefficients of CRMPs and microfilaments were significantly higher than those of microtubules. Immunoprecipitation assay confirmed that CRMPs could bind to microtubules and microfilaments, and that the expression of CRMP5 in neurons increased with the increase of P region, and the expression of microtubules and microfilaments decreased when P region increased most obviously. The decrease was also most obvious in P region. Conclusion: 1) gradient sucrose centrifugation can separate different components of brain tissue according to the size of particles, and the components in the upper layer of the obtained isolate are identified as growth cones. CRMPs coexisted with microtubules and microfilaments in hippocampal tissue. CRMPs can bind to microtubules and microfilaments in vivo and affect the expression and distribution of microtubules and microfilaments. CRMPs may regulate the growth of neuronal processes by mediating the interaction of cytoskeleton.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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