發(fā)布時(shí)間：2018-04-03 05:15

  本文選題：CIITA　切入點(diǎn)：脫乙酰酶　出處：《南京醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:動(dòng)脈粥樣硬化是一種涉及多因子的病理生理過(guò)程。其中,巨噬細(xì)胞介導(dǎo)的慢性炎癥反應(yīng)和平滑肌細(xì)胞介導(dǎo)的血管重塑是兩個(gè)重要事件。在分子水平上,主要組織相容性復(fù)合物II類(lèi)(MHC II)反式作用因子(CIITA)是這兩個(gè)事件的重要調(diào)節(jié)因子,其轉(zhuǎn)錄調(diào)控IFN-γ對(duì)MHC II的激活作用和I型膠原(COL1A2)基因的抑制作用。CIITA的轉(zhuǎn)錄活性受到多種因素影響,包括啟動(dòng)子的選擇,mRNA的穩(wěn)定性以及翻譯后修飾,其中翻譯后修飾(Post-translational modification, PTM,包括甲基化、磷酸化、乙�；�)調(diào)控通過(guò)調(diào)節(jié)CIITA的亞細(xì)胞定位、蛋白質(zhì)穩(wěn)定性等多種的機(jī)制來(lái)調(diào)控CIITA的轉(zhuǎn)錄活性,是近年來(lái)的一個(gè)研究熱點(diǎn)。因此,我們進(jìn)一步研究了脫乙酰酶(HDAC2及SIRT1)介導(dǎo)的賴(lài)氨酸去乙�；瘜�(duì)CIITA轉(zhuǎn)錄活性的調(diào)節(jié)及其在動(dòng)脈粥樣硬化中的病理生理學(xué)意義。 方法:我們?cè)诰奘杉?xì)胞和平滑肌細(xì)胞中通過(guò)免疫共沉淀分析檢測(cè)CIITA和脫乙酰酶(HDAC2及SIRT1)的互相作用。在培養(yǎng)的細(xì)胞中共表達(dá)脫乙酰酶(HDAC2及SIRT1)和CIITA,或給予TSA處理抑制HDAC2活性、白藜蘆醇處理激活SIRT1活性及煙酰胺處理抑制SIRT1活性,或過(guò)表達(dá)siHDAC2干擾HDAC2合成,再運(yùn)用免疫沉淀結(jié)合Western Blotting分析CIITA乙�；降淖兓＝又�,我們通過(guò)熒光素酶報(bào)告基因活性分析實(shí)驗(yàn)檢測(cè)脫乙酰酶(HDAC2及SIRT1)是否影響了CIITA靶基因(MHC II及COL1A2)啟動(dòng)子的活性,并通過(guò)實(shí)時(shí)定量PCR檢測(cè)脫乙酰酶對(duì)CIITA靶基因(MHC II及COL1A2)mRNA表達(dá)的影響。為了進(jìn)一步分析脫乙酰酶調(diào)控CIITA轉(zhuǎn)錄活性的機(jī)制,我們運(yùn)用染色質(zhì)免疫沉淀檢測(cè)HDAC2及其抑制劑對(duì)CIITA與其靶基因啟動(dòng)子結(jié)合的影響。最后,我們探討了HDAC2對(duì)CIITA穩(wěn)定性的影響。在培養(yǎng)的細(xì)胞中給予TSA處理或過(guò)表達(dá)siHDAC2,同時(shí)給予蛋白酶體抑制劑MG132處理,運(yùn)用Western blotting分析CIITA蛋白降解的變化;并運(yùn)用染色質(zhì)免疫沉淀結(jié)合定量PCR檢測(cè)CIITA與靶基因啟動(dòng)子結(jié)合的情況。 結(jié)果:在平滑肌細(xì)胞和巨噬細(xì)胞中HDAC2和CIITA互相作用,并能夠特異性去乙�；疌IITA;HDAC2通過(guò)蛋白酶體作用促進(jìn)CIITA的降解,從而抑制了CIITA靶基因啟動(dòng)子的活性、降低了CIITA與靶基因啟動(dòng)子的結(jié)合及CIITA靶基因mRNA的水平。另一方面,在平滑肌細(xì)胞和巨噬細(xì)胞中SIRT1和CIITA互相作用并能促使其去乙�；�;SIRT1增強(qiáng)了CIITA靶基因啟動(dòng)子的活性及CIITA靶基因mRNA的水平。 結(jié)論:脫乙酰酶(HDAC2及SIRT1)在巨噬細(xì)胞和平滑肌細(xì)胞中調(diào)節(jié)了CIITA的轉(zhuǎn)錄活性,靶向作用脫乙酰酶可能成為潛在的抗動(dòng)脈粥樣硬化治療的策略。因此,我們的研究為臨床上對(duì)動(dòng)脈粥樣硬化進(jìn)行干預(yù)提供了潛在的靶位點(diǎn)。
[Abstract]:Objective: atherosclerosis is a multifactorial pathophysiological process.Among them, macrophage mediated chronic inflammation and smooth muscle cell mediated vascular remodeling are two important events.At the molecular level, the major histocompatibility complex class II MHC II trans-action factor (CIITAA) is an important regulator of these two events.Its transcriptional regulation IFN- 緯 activates MHC II and inhibits the type I collagenous COL1A2) gene. CIITA's transcriptional activity is affected by many factors, including the stability of promoter selector mRNA and posttranslational modification.Post-translational modification (PTM, including methylation, phosphorylation, acetylation, etc.) regulates the transcriptional activity of CIITA by regulating its subcellular localization and protein stability.Therefore, we further studied the role of deacetylase HDAC2 and SIRT1 in the regulation of CIITA transcriptional activity and its pathophysiological significance in atherosclerosis.Methods: the interaction of CIITA with deacetylase HDAC2 and SIRT1 was detected by immunoprecipitation in macrophages and smooth muscle cells.In cultured cells, deacetylase HDAC2 and SIRT1) and CIITAwere co-expressed, or TSA treatment inhibited HDAC2 activity, resveratrol activated SIRT1 activity and nicotinamide inhibited SIRT1 activity, or overexpression of siHDAC2 interfered with HDAC2 synthesis.The changes of CIITA acetylation level were analyzed by immunoprecipitation and Western Blotting.Then we detected whether the activity of deacetylase HDAC2 and SIRT1 affected the activity of CIITA target gene MHCII and COL1A2) by luciferase reporter gene activity analysis.The effect of deacetylase on the expression of CIITA target gene MHCII and COL1A2)mRNA was detected by real-time quantitative PCR.In order to further analyze the mechanism of deacetylase regulating CIITA transcriptional activity, we used chromatin immunoprecipitation to detect the effect of HDAC2 and its inhibitors on the binding of CIITA to its target gene promoter.Finally, we discuss the influence of HDAC2 on the stability of CIITA.The cells were treated with TSA or overexpression of siHDAC2, and treated with proteasome inhibitor MG132. The changes of CIITA protein degradation were analyzed by Western blotting.Chromatin immunoprecipitation and quantitative PCR were used to detect the binding of CIITA to target gene promoter.Results: the interaction of HDAC2 and CIITA in smooth muscle cells and macrophages, and the specific deacetylation of CIITA HDAC2 promoted the degradation of CIITA by proteasome, which inhibited the activity of CIITA target gene promoter.The binding of CIITA to target gene promoter and the level of CIITA target gene mRNA were decreased.On the other hand, SIRT1 and CIITA interacted with CIITA in smooth muscle cells and macrophages, and the deacetylation of SIRT1 enhanced the activity of CIITA target gene promoter and the level of CIITA target gene mRNA.Conclusion: deacetylase (HDAC2 and SIRT1) regulates the transcriptional activity of CIITA in macrophages and smooth muscle cells. Targeted deacetylase may be a potential anti-atherosclerosis therapy strategy.Therefore, our study provides a potential target for clinical intervention in atherosclerosis.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前5條

1 楊菊紅;SIRT1在高糖培養(yǎng)人臍靜脈血管內(nèi)皮細(xì)胞損傷中的作用[D];天津醫(yī)科大學(xué);2009年

2 鄭偉;hSIR2/SIRT1降低蒽環(huán)類(lèi)抗腫瘤藥多柔比星的心臟毒性[D];中國(guó)協(xié)和醫(yī)科大學(xué);2009年

3 袁瓊;二甲基精氨酸—二甲胺水解酶對(duì)內(nèi)皮祖細(xì)胞衰老的調(diào)控與糖尿病血管功能障礙[D];中南大學(xué);2010年

4 曹春雨;熱應(yīng)激細(xì)胞中CIITA基因的表觀遺傳調(diào)控研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

5 夏駿;肺部免疫反應(yīng)和血管重塑中重要轉(zhuǎn)錄事件調(diào)控機(jī)制的研究[D];南京醫(yī)科大學(xué);2013年

相關(guān)碩士學(xué)位論文 前4條

1 張維軍;血管緊張素-（1-7）對(duì)血管緊張素Ⅱ致人臍靜脈內(nèi)皮細(xì)胞衰老的影響及機(jī)制分析[D];山西醫(yī)科大學(xué);2011年

2 張海寧;野生獼猴桃酒生產(chǎn)工藝研究[D];河南科技大學(xué);2011年

3 王楠;高糖對(duì)人臍靜脈內(nèi)皮細(xì)胞NO分泌及SIRT1表達(dá)的影響[D];天津醫(yī)科大學(xué);2009年

4 趙宇豪;1、SIRT1在CIITA介導(dǎo)MHC Ⅱ轉(zhuǎn)錄激活中的作用 2、組蛋白甲基化轉(zhuǎn)移酶SUV39H2在心梗中的作用機(jī)制初探[D];南京醫(yī)科大學(xué);2012年

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本文編號(hào)：1703781