脱乙酰酶在巨噬细胞和平滑肌细胞中对CIITA转录活性的调节

发布时间：2018-04-03 05:15

本文选题：CIITA　切入点：脱乙酰酶　出处：《南京医科大学》2010年硕士论文

【摘要】： 目的:动脉粥样硬化是一种涉及多因子的病理生理过程。其中,巨噬细胞介导的慢性炎症反应和平滑肌细胞介导的血管重塑是两个重要事件。在分子水平上,主要组织相容性复合物II类(MHC II)反式作用因子(CIITA)是这两个事件的重要调节因子,其转录调控IFN-γ对MHC II的激活作用和I型胶原(COL1A2)基因的抑制作用。CIITA的转录活性受到多种因素影响,包括启动子的选择,mRNA的稳定性以及翻译后修饰,其中翻译后修饰(Post-translational modification, PTM,包括甲基化、磷酸化、乙酰化等)调控通过调节CIITA的亚细胞定位、蛋白质稳定性等多种的机制来调控CIITA的转录活性,是近年来的一个研究热点。因此,我们进一步研究了脱乙酰酶(HDAC2及SIRT1)介导的赖氨酸去乙酰化对CIITA转录活性的调节及其在动脉粥样硬化中的病理生理学意义。方法:我们在巨噬细胞和平滑肌细胞中通过免疫共沉淀分析检测CIITA和脱乙酰酶(HDAC2及SIRT1)的互相作用。在培养的细胞中共表达脱乙酰酶(HDAC2及SIRT1)和CIITA,或给予TSA处理抑制HDAC2活性、白藜芦醇处理激活SIRT1活性及烟酰胺处理抑制SIRT1活性,或过表达siHDAC2干扰HDAC2合成,再运用免疫沉淀结合Western Blotting分析CIITA乙酰化水平的变化。接着,我们通过荧光素酶报告基因活性分析实验检测脱乙酰酶(HDAC2及SIRT1)是否影响了CIITA靶基因(MHC II及COL1A2)启动子的活性,并通过实时定量PCR检测脱乙酰酶对CIITA靶基因(MHC II及COL1A2)mRNA表达的影响。为了进一步分析脱乙酰酶调控CIITA转录活性的机制,我们运用染色质免疫沉淀检测HDAC2及其抑制剂对CIITA与其靶基因启动子结合的影响。最后,我们探讨了HDAC2对CIITA稳定性的影响。在培养的细胞中给予TSA处理或过表达siHDAC2,同时给予蛋白酶体抑制剂MG132处理,运用Western blotting分析CIITA蛋白降解的变化;并运用染色质免疫沉淀结合定量PCR检测CIITA与靶基因启动子结合的情况。结果:在平滑肌细胞和巨噬细胞中HDAC2和CIITA互相作用,并能够特异性去乙酰化CIITA;HDAC2通过蛋白酶体作用促进CIITA的降解,从而抑制了CIITA靶基因启动子的活性、降低了CIITA与靶基因启动子的结合及CIITA靶基因mRNA的水平。另一方面,在平滑肌细胞和巨噬细胞中SIRT1和CIITA互相作用并能促使其去乙酰化;SIRT1增强了CIITA靶基因启动子的活性及CIITA靶基因mRNA的水平。结论:脱乙酰酶(HDAC2及SIRT1)在巨噬细胞和平滑肌细胞中调节了CIITA的转录活性,靶向作用脱乙酰酶可能成为潜在的抗动脉粥样硬化治疗的策略。因此,我们的研究为临床上对动脉粥样硬化进行干预提供了潜在的靶位点。
[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- 纬 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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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