β-arrestin-1敲除小鼠中缩胆囊素对胰岛β细胞的作用及机制

发布时间：2018-01-19 02:01

本文关键词： arrestin 基因敲除小鼠缩胆囊素胰岛　出处：《山东大学》2016年硕士论文　论文类型：学位论文

【摘要】：Arrestins是一组具有重要作用的蛋白。它可调节G蛋白偶联受体的下游信号转导。Arrestin同G蛋白偶联受体的结合使G蛋白介导的信号通路被阻断,且介导G蛋白偶联受体的内吞；Arrestin还启动新的非G蛋白依赖的信号途径,例如ERK和Src等信号途径。除了结合GPCR, arrestin可以同许多细胞表面受体家族及多种信号途径蛋白结合。哺乳动物表达arrestin的四种亚型,其中P-arrestin-1和P-arrestin-2的表达在哺乳动物中十分广泛,并且具有十分重要的功能。为了研究β-arrestin-1和β-arrestin-2的生理功能,β-arrestin-1和β-arrestin-2两种基因敲除小鼠被广泛应用。目前,应用β-arrestin-1和β-arrestin-2的基因敲除小鼠,研究许多生理问题：G蛋白偶联受体受到相关激素或配体刺激后,下游信号的转导和调控；各系统器官的发育及功能的维持；各种疾病及外界环境刺激所致疾病的转归等。其中较多的研究结果来自于β-arrestin-2基因敲除小鼠,而对β-arrestin-1基因敲除小鼠的研究较少。本实验室的前期胰岛p细胞系MIN6细胞实验表明,β-arrestin-1在硫化缩胆囊素八肽(CCK-8s)抗p细胞凋亡中发挥着重要作用；为了研究CCK-8s对原代胰岛p细胞的作用,我们通过β-arrestin-1的敲除小鼠的胰岛和胰岛p细胞,证实了β-arrestin-1在CCK-8s结合CCK1受体进而促进胰岛素分泌及抗胰岛p细胞凋亡中的作用。研究胰岛p细胞中CCK1受体激活后不同下游通路的偏向性,对开发新的抗糖尿病药物奠定了基础。[研究目的]运用β-arrestin-1敲除小鼠,研究CCK-8s对胰岛p细胞凋亡和胰岛素分泌的作用及机制。[研究方法]1.实验用基因敲除小鼠及野生型小鼠的准备(1) 纯合敲基因小鼠同C57小鼠的回交。(2) F1代杂合小鼠的选育及配种。(3) 逆转录PCR区分野生型及非野生型F2代小鼠。(4) 实时定量PCR区分F2代杂合及纯合小鼠。(5) 以上述方法扩大种群数量,选取同窝野生及纯合基因敲除小鼠进行动物实验。2.动物实验(1) 使用ELISA试剂盒检钡β-arrestin-1敲除小鼠和野生C57小鼠胰岛给予CCK-8s后胰岛素分泌的变化。(2) 使用ELISA试剂盒检测β-arrestin-1敲除小鼠和野生C57小鼠胰岛给予CCK-8s后IP3, cAMP的变化。(3) P-arrestin-1敲除小鼠和野生C57小鼠STZ糖尿病造模,期间给予CCK-8s,验证β-arrestin-1同CCK-8s保护作用是否相关。(4) 用蛋白印记分析技术(Western blot)检测β-arrestin-1敲除小鼠和野生C57小鼠胰岛抗凋亡途径。[研究结果]P-arrestin-1基因敲除小鼠的胰岛同野生型小鼠胰岛相比,在受到CCK-8s刺激后,分泌胰岛素的量减少。P-arrestin-1基因敲除小鼠的胰岛同野生型小鼠胰岛相比,CCK-8s通过激活CCKI受体引起cAMP的累积效应下降。CCK-8能改善STZ糖尿病造模的C57野生型小鼠的葡萄糖耐量,但不能改善β-arrestin-1敲除小鼠经STZ糖尿病造模后的糖耐量。Western blot显示,β-arrestin-1基因敲除小鼠的胰岛同野生型小鼠胰岛相比,CCK-8s引起p90RSK-phosphor-Bad信号通路的激活被明显抑制。[结论]β-arrestin-1在CCK-8s通过CCK1受体所引起的胰岛p细胞胰岛素的分泌和对p细胞抗凋亡过程中具有十分重要的作用。
[Abstract]:Arrestins is a group which plays an important role in the signaling pathway downstream signal transduction proteins..Arrestin it can regulate G protein coupled receptor and G protein coupled receptor binding to the G protein mediated endocytosis is blocked, and mediated by G protein coupled receptor; Arrestin signaling pathway also launched new non G dependent protein. For example, ERK and Src signaling pathway. In addition with GPCR, arrestin can be combined with a protein surface multicellular receptor family and multiple signaling pathways. Mammalian expression of four subtypes of arrestin, P-arrestin-1 and P-arrestin-2 expression in mammals is very extensive, and has very important function. In order to study the physiological functions of -arrestin-1 and beta -arrestin-2 beta, beta -arrestin-1 and beta -arrestin-2 two gene knockout mice are widely used. At present, the application of beta -arrestin-1 and beta -arrestin-2 gene knockout mice, research Many physical problems: G protein coupled receptors are related to hormone or ligand stimulation, signal transduction and regulation of downstream; maintain the development and function of organs and systems; a variety of diseases and prognosis of diseases caused by external environmental stimuli. Many research results from the beta -arrestin-2 gene knockout mice, while the beta -arrestin-1 gene knockout mice are seldom studied. That the pancreatic P cell line MIN6 cells in our laboratory, beta -arrestin-1 in curing cholecystokinin eight peptide (CCK-8s) plays an important role in anti apoptosis of P cells; in order to study the effects of CCK-8s on cultured islet P cells, we through beta -arrestin-1 knockout mice islet and islet P cells, confirmed the beta -arrestin-1 in CCK-8s binding CCK1 receptor and promote the secretion and anti apoptosis of P cells in insulin action. CCK1 study of pancreatic islet P cell receptor kinase The bias of different downstream pathways of the work, laid the foundation for research purposes. The use of beta -arrestin-1 knockout mice to develop new anti diabetic drugs, gene knockout mice and wild-type mice. Methods to study the effects and mechanisms of CCK-8s on apoptosis and insulin secretion of islet P cells in the]1. experiment (1) pure combined knock-out mice with C57 mouse backcross. (2) F1 heterozygous mice breeding and breeding. (3) reverse transcriptase PCR distinguish between wild-type and non wild type F2 mice. (4) real time quantitative PCR to distinguish F2 generation heterozygous and homozygous mice. (5) the above method to expand the population the number of selected littermate wild and homozygous knockout mice by animal experiment animal experiment of.2. (1) ELISA kit for detecting beta -arrestin-1 knockout mice islet insulin secretion changes of mice and wild C57 after administration of CCK-8s. (2) using the ELISA kit for detection of beta -arrestin-1 Knockout mice and wild mice islet C57 after administration of CCK-8s IP3, cAMP. (3) P-arrestin-1 knockout mice in diabetic STZ mice and wild C57 model, given during CCK-8s, verify the protective effect of CCK-8s beta -arrestin-1 with correlation. (4) by Western blot analysis technology (Western blot) to detect beta -arrestin-1 knockout mice and wild mice islet C57 anti apoptosis pathway. The results of]P-arrestin-1 gene knockout mice with pancreatic islet compared to wild-type mice, under CCK-8s stimulation of insulin secretion decreased in.P-arrestin-1 knockout mice and wild-type mice compared to pancreatic islet, the cumulative effect of CCK-8s through activation of CCKI receptor causes a decrease in the cAMP C57 wild type mice.CCK-8 STZ diabetes model can improve the glucose tolerance, but cannot improve beta -arrestin-1 knockout mice by STZ diabetic rats after glucose tolerance.Western bl Ot showed that the beta -arrestin-1 gene knockout mice and wild-type mice compared to pancreatic islet, the activation of the p90RSK-phosphor-Bad signaling pathway was inhibited. Conclusion] beta -arrestin-1 in CCK-8s through plays an important role on P secretion and cell apoptosis of CCK1 induced by insulin receptor of islet P cells induced by CCK-8s.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

【相似文献】