發(fā)布時(shí)間：2018-06-17 09:38

  本文選題：SIRT1 + HMGB1　； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：糖尿病視網(wǎng)膜病變是糖尿病最常見(jiàn)的微血管病,也是成人常見(jiàn)的致盲眼病。糖尿病的發(fā)生發(fā)展從視網(wǎng)膜微血管病變,微血管滲漏,出現(xiàn)視網(wǎng)膜缺血缺氧,造成大量無(wú)灌注區(qū)形成,最后形成視網(wǎng)膜新生血管的增值性病變,導(dǎo)致視力嚴(yán)重下降。DR的發(fā)病機(jī)制復(fù)雜,包括多元醇通路流量增加、蛋白激酶C激活、己糖胺途徑流量增多等途徑,以及晚期糖基化終末產(chǎn)物增加,晚期糖基化終產(chǎn)物(AGEs) AGEs積聚在糖尿病視網(wǎng)膜血管細(xì)胞、神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞導(dǎo)致DR的發(fā)病。第一部分AGEs干預(yù)后的ARPE-19細(xì)胞中HMBG1和SIRT1的表達(dá)目的：通過(guò)檢測(cè)AGEs干預(yù)下的ARPE-19細(xì)胞中HMGB1、SIRT1的表達(dá),探討其在DR發(fā)病機(jī)制中的作用方法：AGEs與ARPE-19細(xì)胞共同孵育4,8,12小時(shí)后PCR檢測(cè)細(xì)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。結(jié)果：與對(duì)照組相比實(shí)驗(yàn)組炎性細(xì)胞因子白細(xì)胞介素1、白細(xì)胞介素6以及腫瘤壞死因子mRNA表達(dá)上調(diào),兩組間比較差異有統(tǒng)計(jì)學(xué)意義,并且隨著AGE劑量的增加,兩組間差異更加顯著；與對(duì)照組相比實(shí)驗(yàn)組HMGB1mRNA及蛋白表達(dá)上調(diào),兩組間HBMG1mRNA表達(dá)差異有統(tǒng)計(jì)學(xué)意義；與對(duì)照組相比實(shí)驗(yàn)組SIRT1mRNA、蛋白水平及活性下調(diào),兩組間表達(dá)差異有統(tǒng)計(jì)學(xué)意義。結(jié)論：結(jié)果表明,應(yīng)用AGEs作用后促進(jìn)了TNF-α, IL-1, IL-6、MCP-1、RANTES和IP-lOmRNA表達(dá),呈劑量依賴。AEGs治療也顯著促進(jìn)HMGB1在mRNA和蛋白水平的表達(dá),呈劑量依賴性,同時(shí)降低了SIRT1的活性和蛋白表達(dá)。第二部分HMGB1基因下調(diào)對(duì)AGEs誘導(dǎo)的炎性細(xì)胞因子和趨化因子的影響目的：構(gòu)建慢病介導(dǎo)的短發(fā)夾RNA下調(diào)HMGB1的表達(dá),觀察其對(duì)AGEs介導(dǎo)的TNF-α,IL-1,IL-6、MCP-1、RANTES和IP-10的影響方法：設(shè)計(jì)并合成可抑制HMGB1表達(dá)的sh RNA,轉(zhuǎn)染進(jìn)入不同條件下的ARPE-19細(xì)胞。利用RT-real-time PCR方法檢測(cè)TNF-α, IL-1, IL-6、MCP-1、RANTES和IP-lOmRNA的表達(dá)情況。結(jié)果：與空白對(duì)照組比較,外源性HMGB1能顯著提高IL-1β mRNA的表達(dá)。外源性HMGB1組,敲除HMGB1對(duì)IL-Iβ表達(dá)無(wú)統(tǒng)計(jì)學(xué)差異。在AGEs組,敲除HMGB1能顯著降低IL-1 β mRNA的表達(dá)。對(duì)白細(xì)胞介素6、腫瘤壞死因子及趨化因子的檢測(cè)也得出相同結(jié)果。結(jié)論：下調(diào)HMGB1的表達(dá)能介導(dǎo)AGE誘導(dǎo)的炎性細(xì)抑制胞和趨化因子的表達(dá)第三部分SIRT1激活劑與抑制劑對(duì)HMGB1的影響目的：了解在AGE誘導(dǎo)的ARPE-19中炎性反應(yīng)中SIRT1和HMGB1的關(guān)聯(lián)。方法：通過(guò)SIRT1的激活劑和抑制劑來(lái)調(diào)節(jié)SIRT1的活性。分別用RSA(即白藜蘆醇,SIRT1激活劑)和Sirtinol(SIRT1抑制劑)作用AGE干預(yù)的ARPE-19細(xì)胞,然后做PCR/Western blot分析。結(jié)果：RSA和Sirtinol對(duì)AGE作用的ARPE-19細(xì)胞SIRT1、HMGB1mRNA水平表達(dá)無(wú)影響。sirtinol激活了AGE誘導(dǎo)IL-1和IL-6mRNA的表達(dá),而RSV則抑制了二者的表達(dá)。Sirtinol增加了胞漿HMGB1,但抑制了細(xì)胞核HMGB1的蛋白水平,RSV則是抑制了細(xì)胞質(zhì)HMGB1蛋白水平,增加了細(xì)胞核HMGB1蛋白水平。結(jié)論：SIRT1可能通過(guò)抑制HMGB1從胞漿到細(xì)胞核的遷移和釋放,調(diào)節(jié)AGE誘導(dǎo)的致炎細(xì)胞因子和趨化因子。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2;R774.1

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本文編號(hào)：2030578