神经突起诱向因子（Netrin-4）受体的初步研究

发布时间：2018-07-31 13:14

【摘要】：目的：神经突起生长诱向因子Netrin是一种分泌蛋白，在神经发育所需的轴突诱向及细胞迁移中发挥双重诱向功能——吸引或排斥，主要依赖于生长锥所表达的不同受体结直肠癌缺失蛋白(DCC)或UNC5同源物(UNC5H)，从而传递不同信息。本课题组在从事4～6月孕龄人胎肝cDNA文库大规模测序中发现一种新基因，经生物信息学分析确认为Netrin家族成员，即Netrin-4，已有研究证明Netrin-4具有轴突诱向功能，但尚未明确其受体及信号转导通路。本研究探讨了神经突起生长诱向因子Netrin-4的受体以及Netrin-1已知受体DCC和UNC5H1作为Netrin-4受体的可能性，为从蛋白水平研究其信号转导机制及生物学功能提供依据。方法：构建真核表达载体AP4-Netrin-4、AP4-Netrin-1，转染COS7细胞以获得含有碱性磷酸酶(AP)的融合蛋白。改良Koh法分离、原代培养出生12h内清洁级Wistar乳鼠的嗅球及皮层神经元，采用免疫细胞化学方法鉴定其纯度。AP4-Netrin-4融合蛋白与原代培养第7d的神经元孵育，以亲和细胞化学法及碱性磷酸酶原位显色间接反映Netrin-4受体在神经元的定位(因为该融合蛋白能和Netrin-4受体在细胞表面发生亲和反应，从而可用碱性磷酸酶显色进行检测)。以AP4-Netrin-4为配体，以编码DCC和UNC5H1的真核表达载体转染COS7细胞为对象，采用亲和细胞化学技术检测DCC、UNC5H1作为Netrin-4受体的可能性。再以原代培养神经元为对象，检测受体结合动力学、受体竞争结合动力学。结果：原代培养第7d的嗅球及皮层神经元胞体饱满，突起长并交织成网状：免疫细胞化学染色见神经丝蛋白(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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341

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