PKR蛋白结合功能促进胰岛β细胞增殖机制研究

发布时间：2018-05-23 16:14

本文选题：PKR + 蛋白结合功能　；参考：《南京医科大学》2015年硕士论文

【摘要】：目的:PKR(dsRNA-dependent protein kinase)是胞内模式识别受体,也是丝/苏蛋白激酶,被发现参与了外周组织的胰岛素抵抗(insulin resistance),和中心组织胰岛β细胞周期阻滞,在调控机体胰岛β细胞整体功能失代偿和2型糖尿病发生过程中具有至关重要的作用。除了激酶活性,PKR还有蛋白结合功能,而其对胰岛β细胞功能的调节并不清楚。本研究旨在探讨PKR蛋白结合功能对胰岛β细胞增殖的分子机制。方法:采用Coumermycin/GyrB-PKR-K296H融合基因截短体体系,在胰岛β细胞(MIN6细胞和原代胰岛)中构建PKR蛋白结合功能上调模型,即在激酶活性未活化的情况下,单因素研究PKR蛋白结合功能。免疫共沉淀(Co-Immunoprecipitation)检测PKR与TRAF2/3/6的蛋白结合变化;免疫印迹检测PKR过表达后其底物eIFα的磷酸化水平。噻唑蓝(MTT)法、EdU荧光标记和流式细胞术分别评价胰岛β细胞的生长活力、细胞增殖及周期进程的变化。Real-time RT-PCR和Western blot实验分别测定细胞周期蛋白cyclin D1、cyclinD2、CDK2、CDK4、c-Myc和p21、p27以及p53的mRNA和蛋白水平。转染si-c-Myc,si-TRAF2,si-TRAF6,si-RIP1的小干扰RNA或给予NFκB抑制剂(Bay11-7082)预处理;核质分离研究细胞核p65的蛋白水平;免疫荧光实验观察NFκB(p65)的亚细胞结构定位;荧光素酶报告基因实验(Luciferase Assay)分析NFκB的转录活性。转染全长PKR-K296R后,辨析糖脂毒性或炎症因子刺激对MIN6细胞PKR蛋白结合功能的影响。结果:Coumermycin/GyrB-PKR-K296H融合基因截短体体系成功构建了胰岛β细胞无激酶活性的PKR蛋白结合功能上调模型;PKR蛋白结合功能可以促进胰岛β细胞生存活力,增殖和G1/S期转换;并能诱导c-Myc的m RNA和蛋白水平显著上调,而对cyclin D1、cyclinD2、CDK2、CDK4和p21、p27以及p53无明显影响;c-Myc的上调依赖于NFκB的转录活性;而PKR蛋白结合功能上调伴随NFκB的活化;si-TRAF2而不是si-TRAF6能显著阻断PKR蛋白结合功能介导的NFκB转录活性、c-myc上调和胰岛β细胞增殖;PKR蛋白结合功能也能诱导RIP1的泛素化并参与TRAF2调控的胰岛β细胞促增殖作用;而PKR蛋白结合功能也能缓解糖脂毒性和炎症因子诱导的胰岛β细胞生长抑制。结论:PKR蛋白结合结合功能能通过促进细胞周期G1/S转化来刺激胰岛β细胞增殖,这与周期相关蛋白c-Myc的表达上调密切联系。NFκB的转录活性参与了PKR蛋白结合功能诱导的c-Myc基因表达;PKR通过募集TRAF2来激活下游信号分子,泛素化RIP1并激活NFκB转录活性。本论文研究揭示了PKR蛋白结合功能通过TRAF2/RIP1/NFκB/c-Myc信号通路,促进胰岛β细胞增殖的新调控机制。结合我们已有的研究,抑制或下调PKR的激酶活性,而保留PKR蛋白结合功能,能促进胰岛β细胞增殖,为改善2型糖尿病的药物治疗提供新的思路。
[Abstract]:Objective: PKRdsRNA-dependent protein kinase, an intracellular pattern recognition receptor and silk / statin kinase, has been found to be involved in insulin resistance in peripheral tissues and in central islet 尾 cell cycle arrest. It plays an important role in regulating the overall functional decompensation of islet 尾 cells and the development of type 2 diabetes. In addition to kinase activity PKR has protein binding function and its regulation on islet 尾-cell function is not clear. The aim of this study was to investigate the molecular mechanism of PKR binding function on the proliferation of islet 尾 cells. Methods: Coumermycin/GyrB-PKR-K296H fusion gene truncation system was used to construct the up-regulation model of PKR protein binding function in islet 尾 -cell line (Mn-6 cells and primary islets). That is to say, without activation of kinase activity, univariate study of PKR protein binding function was carried out. The protein binding between PKR and TRAF2/3/6 was detected by co-immunoprecipitation, and the phosphorylation level of eIF 伪 was detected by Western blot. The growth activity of islet 尾 cells was evaluated by fluorescence labeling and flow cytometry. The changes of cell proliferation and cell cycle progression. Real-time RT-PCR and Western blot assay were used to determine the mRNA and protein levels of cyclin D 1, cyclin D 2, CDK2, CDK4, c Myc, p21 and p27, and p53, respectively. Transfection of si-c-Mycnsi-TRAF2si-TRAF6si-RIP1 or pretreatment with NF 魏 B inhibitor Bay11-7082; nuclear and cytoplasmic separation to study the protein level of nuclear p65; immunofluorescence assay to observe the subcellular structure localization of NF 魏 B p65; luciferase reporter gene assay Luciferase Assayase to analyze the transcriptional activity of NF 魏 B. After transfection of full-length PKR-K296R, the effects of glycolipid toxicity or inflammatory factor stimulation on the binding function of PKR protein in MIN6 cells were analyzed. Results in the truncated body system of the fusion gene of 1: Coumermycin / GyrB-PKR-K296H, a model of up-regulation of PKR protein binding function without kinase activity in islet 尾 cells was successfully constructed, which could promote the viability, proliferation and G 1 / S phase transition of islet 尾 cells. In addition, the m RNA and protein levels of c-Myc were significantly up-regulated, but the up-regulation of cyclin D1cyclin D2CDK2 + CDK4, p21 p27 and p53 was dependent on the transcriptional activity of NF 魏 B. However, the up-regulation of PKR protein binding function associated with the activation of NF 魏 B, and not si-TRAF6, could significantly block the up-regulation of NF 魏 B transcriptional activity mediated by PKR protein binding function and the proliferation of islet 尾 cells. It could also induce the Ubiquigenation of RIP1. And TRAF2 induced proliferation of islet 尾 cells; The binding function of PKR protein also alleviated the growth inhibition of islet 尾 cells induced by glycolipid toxicity and inflammatory factors. Conclusion the cell cycle G1 / S transformation can stimulate the proliferation of islet 尾 cells by the binding function of the protein: PKR. This is closely related to the up-regulation of cyclin c-Myc expression. NF 魏 B is involved in the expression of c-Myc gene induced by PKR protein binding function. PKR activates downstream signaling molecules by recruiting TRAF2, Ubiquitin RIP1 and NF 魏 B transcriptional activity. This study revealed a new regulatory mechanism of PKR protein binding through the TRAF2/RIP1/NF 魏 B/c-Myc signaling pathway to promote the proliferation of islet 尾 cells. In combination with our previous studies, inhibition or down-regulation of PKR kinase activity and preservation of PKR protein binding function can promote the proliferation of islet 尾 cells and provide new ideas for the improvement of drug therapy for type 2 diabetes.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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