GPR50调控β-淀粉样蛋白产生的机制研究

发布时间：2018-11-18 07:15

【摘要】：研究背景和研究目的：阿尔茨海默病(Alzheimer,s disease，AD)是一种常见的神经退行性疾病，临床主要表现为记忆、认知功能障碍。AD在病理学表现主要包括神经细胞内的神经纤维缠结和细胞外淀粉样斑。淀粉样斑的主要成分是β-淀粉样蛋白（Aβ）的肽类物质在细胞外沉积。Aβ是由β淀粉样前体蛋白(Amyloid Precursor Protein，APP)经β-分泌酶和γ-分泌酶作用，产生一系列长短不等的Aβ和AICD。Aβ能够引起神经棘突丢失、神经可塑性抑制、细胞凋亡等，因此，抑制或减少Aβ的产生是目前AD治疗的一个重要方向。 β-分泌酶是淀粉样途径的主要限速酶，BACE1作为(beta-site APP-cleavingenzyme1)主要限速酶，在Aβ产生过程中的起关键作用，在大脑中高度表达。研究表明在AD患者脑中BACE1水平以及活性明显升高。所以如何调控BACE1以及减少Aβ的产生是AD研究领域的一个研究热点。近年越来越多的研究表明，G蛋白偶联受体（GPCR）在AD的病理过程中起着重要作用。可以通过调控α-、β-和γ-分泌酶的活性来调节脑内APP的代谢，进而影响Aβ产生。GPR50作为GPCRs家族一员，是一种G蛋白偶联孤独受体，GPR50在人脑切片海马齿状核位置有表达，与神经元NOGO-A相互作用促进神经轴突生长，而NOGO家族分子在Aβ产生中起着重要作用。结合以上研究背景我们推测GPR50可能参与AD的发病机制，此研究探讨GPR50对β-淀粉样蛋白产生的作用机制，为AD的药物研发以及诊疗提供科学依据。研究内容： 1、GPR50对Aβ产生影响以及机制。 2、GPR50对BACE1影响以及作用机制。方法： 1、细胞存活率的检测：利用MTT比色法检测细胞存活率。 2、GPR50、BACE1、APP在人脑或细胞的分布以及共定位，采用免疫荧光技术结合显微镜技术检测。 3、GPR50mRNA、BACE1mRNA的水平表达：利用RT-PCR和琼脂糖凝胶电泳相结合检测。 4、细胞外Aβ40、Aβ42浓度检测：利用ELISA方法参照说明书进行检测。 5、蛋白表达水平检测：使用Western blot法检测GPR50、BACE1、APP、CTFs、以及溶酶体等。 6、BACE1活性：依据β-Secretase Activity Assay Kit说明书进行检测。 7、GPR50与BACE1、GPR50与APP蛋白相互关系：采用免疫共沉淀法研究蛋白与蛋白相互关系。结果： 1、GPR50对Aβ产生的调控作用 1.1GPR50抑制Aβ产生和APP的代谢。 1.2GPR50质粒转染进HEK-APP细胞，，抑制Aβ40和Aβ42产生，并且抑制作用表现剂量依赖效应。 1.3在原代培养神经元中，转染GPR50siRNA沉默GPR50表达后Aβ产生明显增加。 1.4GPR50通过调控β-分泌酶抑制Aβ产生。过表达GPR50能够减少β-CTF的表达水平,而α-CTF表达水平无明显改变。 2、GPR50对BACE1的调控作用与机制 2.1GPR50与BACE1共定位并且相互作用。 2.2在HEK-293细胞中过表达GPR50，BACE1活性和蛋白水平明显降低。过表达GPR50不影响HEK-293细胞以及神经元的BACE1mRNA水平表达。 2.3HEK-293细胞用溶酶体抑制剂氯化铵或氯喹处理后，过表达GPR50能够加速BACE1蛋白的降解。 2.4CHO细胞用氯喹预处理后，过表达GPR50-FLAG以及BACE1-HA能够使BACE1和LAMP-1共定位的比率显著性增加；沉默GPR50表达后BACE1和LAMP-1共定位比率下降。结论： GPR50通过抑制BACE1酶活性以及蛋白水平，促进BACE1蛋白转运进溶酶体内和加速蛋白在溶酶体降解来减少Aβ生成，因此GPR50与AD的发病机制有关，可以作为AD治疗的药物靶点。
[Abstract]:Background and purpose of the study: Alzheimer's disease (AD) is a common neurodegenerative disease, and its clinical manifestations are memory and cognitive function. Obstacles. AD in the pathology mainly includes the nerve fiber entanglement and the extracellular starch in the nerve cell The main component of the amyloid plaques is the peptide of the P-amyloid (A-type) in the outside of the cell. The effect of AICD on the loss of the spinous process of the nerve, the inhibition of the plasticity of the nerve, the apoptosis of the cells and so on. Therefore, inhibition or reduction of the generation of A-type is an important part of the current AD treatment Direction. The enzyme is the main speed-limiting enzyme of the starch-like pathway, and BACE1 is the main speed-limiting enzyme of the beta-site APP-cleaning enzyme. It plays a key role in the production of A-enzyme and is in the brain. The expression of BACE1 in the brain of AD patients and the activity of BACE1 in the brain of AD patients. So how to control BACE1 and reduce the generation of A-type is one of the areas of AD research. In recent years, more and more studies have shown that G-protein-coupled receptor (GPCR) has been in the process of AD. It plays an important role in the process. It can regulate the metabolism of APP in the brain by regulating the activity of the P-, The GPR50, as a member of the GPCRs family, is a G-protein-coupled receptor, and the GPR50 is expressed in the dentate nucleus of the hippocampus of the human brain. In combination with the above research background, it is suggested that the GPR50 may be involved in the pathogenesis of AD, this study is to explore the mechanism of action of GPR50 on the amyloid-like protein, and to develop and develop the drug for AD. Diagnosis and treatment providing section Basis of study. Study content: 1, GP R50 impact on A-level and mechanism. 2, GP R50 Effects on BACE1 and Mechanism of Action. Method: 1, fine Detection of cell viability: Cell viability was detected using the MTT assay. 2, GPR50, BACE1, APP were in the human brain or cell The distribution and co-location of GPR50mRNA and BACE1mR were detected by immunofluorescent techniques. Horizontal expression of NA: combined with RT-PCR and agarose gel electrophoresis. A-40, A-42 concentration detection: detection by ELISA method with reference to the specification. 5. Detection of protein expression level: use of the Westin The expression of GPR50, BACE1, APP, CTFs and lysosomes were detected by n-blot. Sex: Test according to the Manual-Secrease Activity Assay Kit instructions. 7, GPR50 vs. (BAC) E1, GPR50 and APP protein cross-correlation System: The relationship between protein and protein was studied by the method of immunoprecipitation. Results: 1. The control effect of GPR50 on A-antigen was 1. 1GPR50 inhibited the production of A and the metabolism of APP. 1. The 2GPR50 plasmid was transfected into HEK-APP cells to inhibit the production of A-antigen 40 and A-antigen 42, and to inhibit the production the expression dose-dependent effect. 1. 3 in primary cultured neurons in that expression of the GPR50 siRNA-silent GPR50, a significant increase in the level of A-antigen was found. The GPR50 is produced by the regulation of the inhibition of A-enzyme by the hormone-secreting enzyme. Over-expression of the GPR50 can reduce the level of expression of the F-CTF, The level of F-CTF expression did not change significantly. The regulation and mechanism of GPR50 on BACE1 2. 1GPR50 co-located with BACE1 and interacts. 2. 2 in HEK-293 The expression of GPR50, BACE1 activity and protein level in the cells was significantly reduced. The overexpression of GPR50 did not affect the HEK-293 cells and the gods. Overexpression of GPR50 could accelerate the degradation of BACE1 protein after the treatment with a lysosome inhibitor of BACE1mRNA. R50-FLAG and BACE1-HA significantly increased the ratio of BACE1 and LAMP-1 co-localization; the ratio of BACE1 and LAMP-1 co-location decreased after silence GPR50 expression. Conclusion: GPR50 is inhibited by inhibition of B
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.16

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