SIRT1和HMGB1在AEGs诱导的视网膜色素上皮细胞炎症反应中的作用研究

发布时间：2018-06-17 09:38

本文选题：SIRT1 + HMGB1　；参考：《武汉大学》2016年博士论文

【摘要】：糖尿病视网膜病变是糖尿病最常见的微血管病,也是成人常见的致盲眼病。糖尿病的发生发展从视网膜微血管病变,微血管渗漏,出现视网膜缺血缺氧,造成大量无灌注区形成,最后形成视网膜新生血管的增值性病变,导致视力严重下降。DR的发病机制复杂,包括多元醇通路流量增加、蛋白激酶C激活、己糖胺途径流量增多等途径,以及晚期糖基化终末产物增加,晚期糖基化终产物(AGEs) AGEs积聚在糖尿病视网膜血管细胞、神经元和神经胶质细胞导致DR的发病。第一部分AGEs干预后的ARPE-19细胞中HMBG1和SIRT1的表达目的：通过检测AGEs干预下的ARPE-19细胞中HMGB1、SIRT1的表达,探讨其在DR发病机制中的作用方法：AGEs与ARPE-19细胞共同孵育4,8,12小时后PCR检测细胞中TNF-α,IL-1,IL-6、MCP-1、RANTES和IP-10以及HMGB1、SIRT1mRNA的水平,24h后western blot分析HMGB1、SIRT1蛋白水平。结果：与对照组相比实验组炎性细胞因子白细胞介素1、白细胞介素6以及肿瘤坏死因子mRNA表达上调,两组间比较差异有统计学意义,并且随着AGE剂量的增加,两组间差异更加显著；与对照组相比实验组HMGB1mRNA及蛋白表达上调,两组间HBMG1mRNA表达差异有统计学意义；与对照组相比实验组SIRT1mRNA、蛋白水平及活性下调,两组间表达差异有统计学意义。结论：结果表明,应用AGEs作用后促进了TNF-α, IL-1, IL-6、MCP-1、RANTES和IP-lOmRNA表达,呈剂量依赖。AEGs治疗也显著促进HMGB1在mRNA和蛋白水平的表达,呈剂量依赖性,同时降低了SIRT1的活性和蛋白表达。第二部分HMGB1基因下调对AGEs诱导的炎性细胞因子和趋化因子的影响目的：构建慢病介导的短发夹RNA下调HMGB1的表达,观察其对AGEs介导的TNF-α,IL-1,IL-6、MCP-1、RANTES和IP-10的影响方法：设计并合成可抑制HMGB1表达的sh RNA,转染进入不同条件下的ARPE-19细胞。利用RT-real-time PCR方法检测TNF-α, IL-1, IL-6、MCP-1、RANTES和IP-lOmRNA的表达情况。结果：与空白对照组比较,外源性HMGB1能显著提高IL-1β mRNA的表达。外源性HMGB1组,敲除HMGB1对IL-Iβ表达无统计学差异。在AGEs组,敲除HMGB1能显著降低IL-1 β mRNA的表达。对白细胞介素6、肿瘤坏死因子及趋化因子的检测也得出相同结果。结论：下调HMGB1的表达能介导AGE诱导的炎性细抑制胞和趋化因子的表达第三部分SIRT1激活剂与抑制剂对HMGB1的影响目的：了解在AGE诱导的ARPE-19中炎性反应中SIRT1和HMGB1的关联。方法：通过SIRT1的激活剂和抑制剂来调节SIRT1的活性。分别用RSA(即白藜芦醇,SIRT1激活剂)和Sirtinol(SIRT1抑制剂)作用AGE干预的ARPE-19细胞,然后做PCR/Western blot分析。结果：RSA和Sirtinol对AGE作用的ARPE-19细胞SIRT1、HMGB1mRNA水平表达无影响。sirtinol激活了AGE诱导IL-1和IL-6mRNA的表达,而RSV则抑制了二者的表达。Sirtinol增加了胞浆HMGB1,但抑制了细胞核HMGB1的蛋白水平,RSV则是抑制了细胞质HMGB1蛋白水平,增加了细胞核HMGB1蛋白水平。结论：SIRT1可能通过抑制HMGB1从胞浆到细胞核的迁移和释放,调节AGE诱导的致炎细胞因子和趋化因子。
[Abstract]:Diabetic retinopathy is the most common microvascular disease of diabetes, and it is also a common blindness eye disease in adults. The development of diabetes is from retinal microvascular lesions, microvascular leakage, retinal ischemia and hypoxia, resulting in a large number of non perfusion areas, and finally the formation of value-added lesions of the retinal neovascularization, resulting in severe visual loss. The pathogenesis of DR is complex, including increased flow of polyol pathway, activation of protein kinase C, increased flow of hexanamine pathway, and the increase of late glycosylation end products. Late glycosylated end products (AGEs) AGEs accumulates in diabetic retinal vascular cells, neurons and glial cells lead to the pathogenesis of DR. The first part AGEs dry The expression of HMBG1 and SIRT1 in the prognosis of ARPE-19 cells: by detecting the expression of HMGB1 and SIRT1 in ARPE-19 cells under the intervention of AGEs, the methods of its action in the pathogenesis of DR are discussed: AGEs and ARPE-19 cells are incubated together for PCR detection cells. Level, after 24h Western blot analysis of HMGB1, SIRT1 protein level. Results: compared with the control group, the inflammatory cytokines interleukin 1, interleukin 6 and tumor necrosis factor mRNA expression were up. The difference between the two groups was statistically significant, and with the increase of AGE dose, the difference between the two groups was more significant; compared with the control group, the difference was more significant. The expression of HMGB1mRNA and protein was up-regulated in the experimental group. The difference of HBMG1mRNA expression between the two groups was statistically significant. Compared with the control group, the protein level and activity of the experimental group were down, and the expression difference between the two groups was statistically significant. Conclusion: the results showed that the application of AGEs promoted the expression of TNF-, IL-1, IL-6, MCP-1, RANTES and IP-lOmRNA. Dose dependent.AEGs therapy also significantly promoted the expression of HMGB1 at mRNA and protein levels, in a dose-dependent manner, and decreased the activity and protein expression of SIRT1. Second the effect of the down regulation of HMGB1 gene on the inflammatory cytokines and chemokines induced by AGEs: to construct the slow disease mediated short hairpin RNA to reduce the expression of HMGB1 and to observe its expression. AGEs mediated TNF- alpha, IL-1, IL-6, MCP-1, RANTES and IP-10: designed and synthesized sh RNA that can inhibit the expression of HMGB1, and transfected into the ARPE-19 cells under different conditions. B1 could significantly increase the expression of IL-1 beta mRNA. There was no significant difference in the expression of IL-I beta in the exogenous HMGB1 group. In the AGEs group, the expression of IL-1 beta mRNA was significantly reduced by knockout HMGB1. The same results were also obtained for the detection of interleukin 6, tumor necrosis factor and chemokine. Conclusion: down-regulation of HMGB1 expression can mediate AGE induced inflammation. Expression of sexual fine suppressor and chemokines third part SIRT1 activator and inhibitor effect on HMGB1 purpose: to understand the association of SIRT1 and HMGB1 in the inflammatory response in ARPE-19 induced by AGE. Methods: the activity of SIRT1 is regulated by activators and inhibitors of SIRT1. RSA (resveratrol, SIRT1 activator) and Sirtinol (SIR) are used respectively. T1 inhibitors action AGE interfered ARPE-19 cells and then PCR/Western blot analysis. Results: RSA and Sirtinol showed no effect on ARPE-19 cell SIRT1, HMGB1mRNA level expression. The protein level of HMGB1, RSV, inhibits the cytoplasmic HMGB1 protein level and increases the level of nuclear HMGB1 protein. Conclusion: SIRT1 may regulate the AGE induced inflammatory cytokines and chemokines by inhibiting the migration and release of HMGB1 from the cytoplasm to the nucleus.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R774.1

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