本文選題：嘌呤能P2X7受體 + 大鼠肝星狀細胞　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:酒精性肝臟疾病是因長期大量過度飲酒所導(dǎo)致的各種肝臟損害性疾病。在ALF發(fā)展過程中,活化的HSC是肝臟產(chǎn)生細胞外基質(zhì)(ECM)的主要靶細胞,乙醇的刺激性代謝物乙醛可使肝星狀細胞活化,進而導(dǎo)致纖維化的發(fā)生。隨著對嘌呤受體研究的逐步深入,人們發(fā)現(xiàn)嘌呤能P2X7受體亞型在纖維化疾病中發(fā)揮著重要作用,并且與肝臟疾病有著密切的聯(lián)系。P2X7受體是嘌呤受體家族中獨特的離子通道型受體,可激活鈣離子通道促進Ca2+內(nèi)流,并可促使組織炎癥反應(yīng)的發(fā)生,進而導(dǎo)致肝臟疾病的發(fā)生。糖原合酶激酶-3β(GSK-3β),是一種絲氨酸/蘇氨酸蛋白激酶,分布廣泛,不僅能調(diào)節(jié)糖代謝反應(yīng),還可以介導(dǎo)各種炎癥反應(yīng)和纖維化的發(fā)生。目前有研究報道,抑制GSK-3β可明顯減少腎纖維化模型中膠原I的形成和炎癥細胞因子的產(chǎn)生。激活P2X7受體可刺激PKC依賴性GSK3途徑的活化,再引起GSK3的磷酸化而發(fā)揮作用。目的:通過建立乙醛誘導(dǎo)的HSC活化模型模擬酒精性肝纖維化離體模型。觀察嘌呤能P2X7受體在HSC中的m RNA和蛋白表達情況,對細胞周期的影響,是否對各種炎癥細胞因子的表達有所影響以及對AKT和ERK1/2的磷酸化水平是否有影響。同時探討P2X7受體對乙醛誘導(dǎo)的HSC活化過程中α-SMA和Collagen I的表達的影響,是否通過PKC-GSK3β信號通路產(chǎn)生作用。為預(yù)防與治療酒精性肝纖維化疾病提供新的靶點。方法:通過200μM乙醛刺激HSC48h建立HSC活化模型進行模擬酒精性肝纖維化離體模型。采用Western blot和實時定量PCR觀察P2X7R的表達情況。采用P2X7R激動劑/抑制劑、RNA干擾技術(shù)處理以及流式細胞儀技術(shù)觀察P2X7受體對細胞周期的影響,采用Western blot和實時定量PCR觀察對炎癥因子(TNF-α,IL-6,IL-18和IL-1β)表達以及對α-SMA和Collagen I的表達的影響,并用Western blot觀察其對PKC-GSK3β信號通路的影響以及對p AKT和p ERK1/2的影響。采用PKC激動劑/PKC抑制劑和GSK3β選擇性抑制劑分別觀察PKC與GSK3β在乙醛誘導(dǎo)的HSC活化過程中對α-SMA和Collagen I的表達的影響。結(jié)果:在乙醛誘導(dǎo)的HSC活化模型中,P2X7R表達較正常組顯著升高。采用P2X7R激動劑Bz ATP可明顯升高細胞周期S期細胞比例,炎癥因子(TNF-α,IL-6,IL-18和IL-1β)和α-SMA和Collagen I的表達。而采用P2X7R抑制劑A438079和沉默P2X7R均可明顯降低細胞周期,炎癥因子以及α-SMA和Collagen的表達。此外,Bz ATP亦可增加PKC和p GSK3β/GSK3β比值,A438079和沉默P2X7R則使PKC表達和p GSK3β/GSK3β比值明顯降低。此外,采用PKC激動劑PMA/抑制劑SC-3088研究發(fā)現(xiàn),PMA可促進GSK3β磷酸化以及促進α-SMA和Collagen I的表達,而SC-3088則相反地抑制GSK3β磷酸化并降低α-SMA和Collagen I的表達。GSK3β選擇性抑制劑TDZD-8可明顯抑制GSK3β磷酸化以及抑制α-SMA和Collagen I的表達。更有趣的是,Bz ATP也可明顯增加p AKT和p ERK1/2的表達,而A438079和沉默P2X7R也可明顯AKT和ERK1/2的磷酸化水平。綜合以上結(jié)果說明,P2X7受體可以通過激活PKC-GSK3β信號通路而促進乙醛介導(dǎo)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575

【相似文獻】

相關(guān)碩士學(xué)位論文 前1條

1 武小娟;嘌呤P2X7受體通過PKC-GSK3β途徑介導(dǎo)乙醛誘導(dǎo)的肝星狀細胞活化的研究[D];安徽醫(yī)科大學(xué);2017年

，

本文編號：2024680