神经突起导向因子NETRIN-4与NNAT的相互作用及其表达相关性研究

发布时间：2018-01-11 18:33

本文关键词：神经突起导向因子NETRIN-4与NNAT的相互作用及其表达相关性研究　出处：《中国人民解放军军事医学科学院》2005年博士论文　论文类型：学位论文

【摘要】：神经突起与其靶细胞之间精确联系的建立是由多种导向因子共同作用完成的。Netrin基因家族是此类因子的代表之一。本研究主要围绕该家族成员之一Netrin-4及其相互作用蛋白NNAT(Neuronatin)的结构、功能和表达变化特征进行系统分析。本研究首先构建Netrin-4及其三个结构域的原核表达载体,用IPTG诱导得到相应的融合蛋白,并进一步采用合成肽制备抗Netrin-4特异性抗体。为了探讨Netrin-4发挥作用的分子基础,利用酵母双杂交技术从人胎脑cDNA文库中筛选并通过GST-pull down等实验鉴定出NNAT是其强相互作用蛋白,且证明Netrin-4的EGF重复结构域是Netrin-4与NNAT相互作用的重要结构。为了阐明功能尚属未知的NNAT蛋白与Netrin-4发生相互作用的意义,将NNAT与GFP融合表达,观察到NNAT蛋白在真核细胞中的表达存在核定位的现象,而流式细胞仪检测结果表明融合蛋白的过表达可导致细胞周期改变。进一步通过对所构建的能够稳定表达NNAT的工程细胞株的研究,发现NNAT蛋白仍然存在核定位和细胞周期阻滞现象。这些结果提示NNAT通过定位到细胞核中发挥其生物学功能,可能与细胞的增殖与分化的控制有关。利用RT-PCR技术检测Netrin-4和Nnat在11种细胞系和原代培养神经元中的表达情况,发现二者仅在神经元中有共表达,且在大脑发育的不同时期中Netrin-4和Nnat mRNA的表达特点不同,大鼠出生前后是Nnat表达的高峰期,Netrin-4出生后表达量变化不大。Netrin-4和Nnat在出生前后的高表达提示二者之间的相互作用可能与该时期神经发育过程及神经可塑性密切相关。综上所述,本研究发现Netrin-4和Nnat都是在大脑发育过程中发挥重要作用的基因。Netrin-4除在发育初期发挥神经突起导向功能外,在大脑发育过程有可能通过与NNAT蛋白相互作用,共同影响突触及特定神经回路的形成。本研究为理解神经发育与分化过程中神经突起导向、神经可塑性的分子机制提供了新的数据。
[Abstract]:The establishment of precise connections between neurites and their target cells is accomplished by the interaction of multiple guiding factors. The. Netrin gene family is one of the representatives of these factors. Etrin-4 and its interacting protein nat (. The structure of Neuronatinin. The function and expression change characteristics were systematically analyzed. In this study, the prokaryotic expression vector of Netrin-4 and its three domains was constructed, and the corresponding fusion protein was obtained by IPTG induction. Furthermore, synthetic peptides were used to prepare specific antibodies against Netrin-4. In order to explore the molecular basis of the role of Netrin-4. Yeast two-hybrid technique was used to screen human fetal brain cDNA library and GST-pull down was used to identify NNAT as a strong interacting protein. It is proved that the EGF repeat domain of Netrin-4 is an important structure for the interaction between Netrin-4 and NNAT. In order to elucidate the unknown function of NNAT protein and Netrin-4. The meaning of interaction. The expression of NNAT protein in eukaryotic cells was observed by fusion expression of NNAT and GFP. The results of flow cytometry showed that the overexpression of the fusion protein could lead to cell cycle changes. Further studies on the constructed engineering cell line which can stably express NNAT were carried out. It was found that NNAT protein still exists nuclear localization and cell cycle arrest. These results suggest that NNAT can play its biological function by localization into the nucleus. RT-PCR technique was used to detect the expression of Netrin-4 and Nnat in 11 cell lines and primary cultured neurons. It was found that there was only coexpression of Netrin-4 and Nnat mRNA in neurons, and the expression characteristics of Netrin-4 and Nnat mRNA were different in different stages of brain development. The peak expression of Nnat was before and after birth in rats. The high expression of Netrin-4 and Nnat before and after birth suggests that the interaction between them may be related to the neural development and neural plasticity. Closely related. To sum up. This study found that both Netrin-4 and Nnat are genes that play an important role in the development of the brain. Netrin-4 plays a role in the neurite guidance function in the early stage of development. It is possible that the formation of synapses and specific neural circuits can be affected by interaction with NNAT proteins during brain development. This study is intended to understand the neurite orientation during neural development and differentiation. The molecular mechanism of neural plasticity provides new data.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2005
【分类号】：Q593.2

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