嘌呤P2X7受体通过PKC-GSK3β途径介导乙醛诱导的肝星状细胞活化的研究

发布时间：2018-06-16 01:53

本文选题：嘌呤能P2X7受体 + 大鼠肝星状细胞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：背景:酒精性肝脏疾病是因长期大量过度饮酒所导致的各种肝脏损害性疾病。在ALF发展过程中,活化的HSC是肝脏产生细胞外基质(ECM)的主要靶细胞,乙醇的刺激性代谢物乙醛可使肝星状细胞活化,进而导致纤维化的发生。随着对嘌呤受体研究的逐步深入,人们发现嘌呤能P2X7受体亚型在纤维化疾病中发挥着重要作用,并且与肝脏疾病有着密切的联系。P2X7受体是嘌呤受体家族中独特的离子通道型受体,可激活钙离子通道促进Ca2+内流,并可促使组织炎症反应的发生,进而导致肝脏疾病的发生。糖原合酶激酶-3β(GSK-3β),是一种丝氨酸/苏氨酸蛋白激酶,分布广泛,不仅能调节糖代谢反应,还可以介导各种炎症反应和纤维化的发生。目前有研究报道,抑制GSK-3β可明显减少肾纤维化模型中胶原I的形成和炎症细胞因子的产生。激活P2X7受体可刺激PKC依赖性GSK3途径的活化,再引起GSK3的磷酸化而发挥作用。目的:通过建立乙醛诱导的HSC活化模型模拟酒精性肝纤维化离体模型。观察嘌呤能P2X7受体在HSC中的m RNA和蛋白表达情况,对细胞周期的影响,是否对各种炎症细胞因子的表达有所影响以及对AKT和ERK1/2的磷酸化水平是否有影响。同时探讨P2X7受体对乙醛诱导的HSC活化过程中α-SMA和Collagen I的表达的影响,是否通过PKC-GSK3β信号通路产生作用。为预防与治疗酒精性肝纤维化疾病提供新的靶点。方法:通过200μM乙醛刺激HSC48h建立HSC活化模型进行模拟酒精性肝纤维化离体模型。采用Western blot和实时定量PCR观察P2X7R的表达情况。采用P2X7R激动剂/抑制剂、RNA干扰技术处理以及流式细胞仪技术观察P2X7受体对细胞周期的影响,采用Western blot和实时定量PCR观察对炎症因子(TNF-α,IL-6,IL-18和IL-1β)表达以及对α-SMA和Collagen I的表达的影响,并用Western blot观察其对PKC-GSK3β信号通路的影响以及对p AKT和p ERK1/2的影响。采用PKC激动剂/PKC抑制剂和GSK3β选择性抑制剂分别观察PKC与GSK3β在乙醛诱导的HSC活化过程中对α-SMA和Collagen I的表达的影响。结果:在乙醛诱导的HSC活化模型中,P2X7R表达较正常组显著升高。采用P2X7R激动剂Bz ATP可明显升高细胞周期S期细胞比例,炎症因子(TNF-α,IL-6,IL-18和IL-1β)和α-SMA和Collagen I的表达。而采用P2X7R抑制剂A438079和沉默P2X7R均可明显降低细胞周期,炎症因子以及α-SMA和Collagen的表达。此外,Bz ATP亦可增加PKC和p GSK3β/GSK3β比值,A438079和沉默P2X7R则使PKC表达和p GSK3β/GSK3β比值明显降低。此外,采用PKC激动剂PMA/抑制剂SC-3088研究发现,PMA可促进GSK3β磷酸化以及促进α-SMA和Collagen I的表达,而SC-3088则相反地抑制GSK3β磷酸化并降低α-SMA和Collagen I的表达。GSK3β选择性抑制剂TDZD-8可明显抑制GSK3β磷酸化以及抑制α-SMA和Collagen I的表达。更有趣的是,Bz ATP也可明显增加p AKT和p ERK1/2的表达,而A438079和沉默P2X7R也可明显AKT和ERK1/2的磷酸化水平。综合以上结果说明,P2X7受体可以通过激活PKC-GSK3β信号通路而促进乙醛介导的HSC-T6的活化。
[Abstract]:Background: alcoholic liver disease is a variety of liver damage caused by excessive drinking over the long term. During the development of ALF, activated HSC is the main target cell for the production of extracellular matrix (ECM) in the liver. The stimulant metabolite of ethanol can make hepatic stellate cells live and lead to the occurrence of fibrosis. It has been found that the purinergic P2X7 receptor subtype plays an important role in fibrotic diseases and has a close relationship with liver diseases. The.P2X7 receptor is a unique ion channel receptor in the purine receptor family, which activates the calcium channel to promote the Ca2+ influx and promotes the occurrence of inflammatory reactions in the tissues. The occurrence of liver disease. Glycogen synthase kinase -3 beta (GSK-3 beta), a serine / threonine protein kinase, is widely distributed. It can not only regulate glucose metabolism, but also mediate the occurrence of various inflammatory reactions and fibrosis. Currently, it is reported that inhibition of GSK-3 beta can significantly reduce the formation of collagen I in the model of renal fibrosis and the fine inflammation. The activation of P2X7 receptor stimulates the activation of the PKC dependent GSK3 pathway and causes the phosphorylation of GSK3. Objective: to simulate the model of alcoholic liver fibrosis in vitro by establishing the HSC activation model induced by acetaldehyde. The expression of M RNA and protein in HSC, and the effect on the cell cycle of the purine P2X7 receptor, is observed. The influence of the expression of various inflammatory cytokines and the effect on the phosphorylation of AKT and ERK1/2, and the effect of P2X7 receptor on the expression of alpha -SMA and Collagen I in the activation of acetaldehyde induced HSC, whether or not the PKC-GSK3 beta signaling pathway is produced, for the prevention and treatment of alcoholic liver fibrosis. Method: a model of alcohol induced liver fibrosis was simulated by a HSC activation model of 200 mu M acetaldehyde to simulate alcoholic liver fibrosis. The expression of P2X7R was observed by Western blot and real-time quantitative PCR. P2X7R agonist / inhibitor, RNA interference technique and flow cytometry were used to observe the effect of P2X7 receptor on the cell cycle. Western blot and real-time quantitative PCR were used to observe the expression of inflammatory factors (TNF-, IL-6, IL-18 and IL-1 beta), and the effect on the expression of alpha and Collagen I. Do not observe the effect of PKC and GSK3 beta on the expression of alpha -SMA and Collagen I during the activation of acetaldehyde induced HSC. Results: in the HSC activation model induced by acetaldehyde, the expression of P2X7R is significantly higher than that in the normal group. The percentage of cell cycle S cells can be significantly increased by the P2X7R agonist Bz ATP. And the expression of Collagen I, and the use of P2X7R inhibitor A438079 and silent P2X7R can significantly reduce the cell cycle, inflammatory factors and the expression of alpha -SMA and Collagen. In addition, Bz ATP can also increase PKC and P GSK3 beta ratio. The study of inhibitor SC-3088 found that PMA can promote the phosphorylation of GSK3 beta and promote the expression of alpha -SMA and Collagen I, while SC-3088 inhibits GSK3 beta phosphorylation and reduces the expression of alpha -SMA and Collagen I. TP can also significantly increase the expression of P AKT and P ERK1/2, while A438079 and silent P2X7R can also clear the phosphorylation level of AKT and ERK1/2. These results suggest that P2X7 receptors can activate the activation of aldehyde mediated HSC-T6 by activating the PKC-GSK3 beta signaling pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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