【摘要】：目的：神經(jīng)突起生長(zhǎng)誘向因子Netrin是一種分泌蛋白，在神經(jīng)發(fā)育所需的軸突誘向及細(xì)胞遷移中發(fā)揮雙重誘向功能——吸引或排斥，主要依賴于生長(zhǎng)錐所表達(dá)的不同受體結(jié)直腸癌缺失蛋白(DCC)或UNC5同源物(UNC5H)，從而傳遞不同信息。本課題組在從事4～6月孕齡人胎肝cDNA文庫大規(guī)模測(cè)序中發(fā)現(xiàn)一種新基因，經(jīng)生物信息學(xué)分析確認(rèn)為Netrin家族成員，即Netrin-4，已有研究證明Netrin-4具有軸突誘向功能，但尚未明確其受體及信號(hào)轉(zhuǎn)導(dǎo)通路。本研究探討了神經(jīng)突起生長(zhǎng)誘向因子Netrin-4的受體以及Netrin-1已知受體DCC和UNC5H1作為Netrin-4受體的可能性，為從蛋白水平研究其信號(hào)轉(zhuǎn)導(dǎo)機(jī)制及生物學(xué)功能提供依據(jù)。方法：構(gòu)建真核表達(dá)載體AP4-Netrin-4、AP4-Netrin-1，轉(zhuǎn)染COS7細(xì)胞以獲得含有堿性磷酸酶(AP)的融合蛋白。改良Koh法分離、原代培養(yǎng)出生12h內(nèi)清潔級(jí)Wistar乳鼠的嗅球及皮層神經(jīng)元，采用免疫細(xì)胞化學(xué)方法鑒定其純度。AP4-Netrin-4融合蛋白與原代培養(yǎng)第7d的神經(jīng)元孵育，以親和細(xì)胞化學(xué)法及堿性磷酸酶原位顯色間接反映Netrin-4受體在神經(jīng)元的定位(因?yàn)樵撊诤系鞍啄芎蚇etrin-4受體在細(xì)胞表面發(fā)生親和反應(yīng)，從而可用堿性磷酸酶顯色進(jìn)行檢測(cè))。以AP4-Netrin-4為配體，以編碼DCC和UNC5H1的真核表達(dá)載體轉(zhuǎn)染COS7細(xì)胞為對(duì)象，采用親和細(xì)胞化學(xué)技術(shù)檢測(cè)DCC、UNC5H1作為Netrin-4受體的可能性。再以原代培養(yǎng)神經(jīng)元為對(duì)象，檢測(cè)受體結(jié)合動(dòng)力學(xué)、受體競(jìng)爭(zhēng)結(jié)合動(dòng)力學(xué)。結(jié)果：原代培養(yǎng)第7d的嗅球及皮層神經(jīng)元胞體飽滿，突起長(zhǎng)并交織成網(wǎng)狀：免疫細(xì)胞化學(xué)染色見神經(jīng)絲蛋白(neurofilament)在神
[Abstract]:Objective: Netrin, a growth inducer of neurite, is a secretory protein, which plays a dual inductive function-attraction or rejection-in axon inducement and cell migration for nerve development. It mainly depends on (DCC) or UNC5 homologues (UNC5H) expressed by different receptors in colorectal cancer, which can transmit different information. A novel gene was found in large scale sequencing of human fetal liver cDNA library from 4 to 6 months of gestational age. It was identified by bioinformatics analysis as a member of Netrin family, i.e. Netrin-4. It has been proved that Netrin-4 has axon inductive function. However, its receptor and signal transduction pathway have not been clarified. This study investigated the receptors of neurite growth-inducing factor (Netrin-4) and the known receptors of Netrin-1 (DCC and UNC5H1) as Netrin-4 receptors, which provided a basis for the study of signal transduction mechanism and biological functions at the protein level. Methods: Eukaryotic expression vector AP4-Netrin-4 AP4-Netrin-1 was constructed and transfected into COS7 cells to obtain the fusion protein containing alkaline phosphatase (AP). The olfactory bulb and cortical neurons of Wistar neonate of clean grade were isolated by modified Koh method and incubated with neurons on the 7th day of primary culture by immunocytochemistry. The localization of Netrin-4 receptor in neurons was indirectly reflected by affinity cytochemistry and in situ coloration of alkaline phosphatase (because the fusion protein could react with Netrin-4 receptor on the cell surface, which could be detected by alkaline phosphatase). Using AP4-Netrin-4 as ligand, the eukaryotic expression vector encoding DCC and UNC5H1 was transfected into COS7 cells. The possibility of DCC-UNC5H1 as Netrin-4 receptor was detected by affinity cytochemistry. The receptor binding kinetics and receptor competitive binding kinetics were determined by primary cultured neurons. Results: on the 7th day of primary culture, the neurons of olfactory bulb and cortex were plump, the processes were long and interwoven into a network. Immunocytochemical staining showed that the neurofilament protein (neurofilament) was present in Shenshen.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R341

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