本文關(guān)鍵詞：神經(jīng)突起導(dǎo)向因子NETRIN-4與NNAT的相互作用及其表達相關(guān)性研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2005年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Natrin-4 酵母雙雜交 Neuronatin 神經(jīng)發(fā)育 突觸形成

【摘要】：神經(jīng)突起與其靶細(xì)胞之間精確聯(lián)系的建立是由多種導(dǎo)向因子共同作用完成的。Netrin基因家族是此類因子的代表之一。本研究主要圍繞該家族成員之一Netrin-4及其相互作用蛋白NNAT(Neuronatin)的結(jié)構(gòu)、功能和表達變化特征進行系統(tǒng)分析。 本研究首先構(gòu)建Netrin-4及其三個結(jié)構(gòu)域的原核表達載體,用IPTG誘導(dǎo)得到相應(yīng)的融合蛋白,并進一步采用合成肽制備抗Netrin-4特異性抗體。為了探討Netrin-4發(fā)揮作用的分子基礎(chǔ),利用酵母雙雜交技術(shù)從人胎腦cDNA文庫中篩選并通過GST-pull down等實驗鑒定出NNAT是其強相互作用蛋白,且證明Netrin-4的EGF重復(fù)結(jié)構(gòu)域是Netrin-4與NNAT相互作用的重要結(jié)構(gòu)。為了闡明功能尚屬未知的NNAT蛋白與Netrin-4發(fā)生相互作用的意義,將NNAT與GFP融合表達,觀察到NNAT蛋白在真核細(xì)胞中的表達存在核定位的現(xiàn)象,而流式細(xì)胞儀檢測結(jié)果表明融合蛋白的過表達可導(dǎo)致細(xì)胞周期改變。進一步通過對所構(gòu)建的能夠穩(wěn)定表達NNAT的工程細(xì)胞株的研究,發(fā)現(xiàn)NNAT蛋白仍然存在核定位和細(xì)胞周期阻滯現(xiàn)象。這些結(jié)果提示NNAT通過定位到細(xì)胞核中發(fā)揮其生物學(xué)功能,可能與細(xì)胞的增殖與分化的控制有關(guān)。利用RT-PCR技術(shù)檢測Netrin-4和Nnat在11種細(xì)胞系和原代培養(yǎng)神經(jīng)元中的表達情況,發(fā)現(xiàn)二者僅在神經(jīng)元中有共表達,且在大腦發(fā)育的不同時期中Netrin-4和Nnat mRNA的表達特點不同,大鼠出生前后是Nnat表達的高峰期,Netrin-4出生后表達量變化不大。Netrin-4和Nnat在出生前后的高表達提示二者之間的相互作用可能與該時期神經(jīng)發(fā)育過程及神經(jīng)可塑性密切相關(guān)。 綜上所述,本研究發(fā)現(xiàn)Netrin-4和Nnat都是在大腦發(fā)育過程中發(fā)揮重要作用的基因。Netrin-4除在發(fā)育初期發(fā)揮神經(jīng)突起導(dǎo)向功能外,在大腦發(fā)育過程有可能通過與NNAT蛋白相互作用,共同影響突觸及特定神經(jīng)回路的形成。本研究為理解神經(jīng)發(fā)育與分化過程中神經(jīng)突起導(dǎo)向、神經(jīng)可塑性的分子機制提供了新的數(shù)據(jù)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：Q593.2

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