低氧诱导内皮细胞Brm表达上调的机制研究

发布时间：2018-05-13 06:50

本文选题：低氧性肺动脉高压 + Brm　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景:低氧性肺动脉高压(Hypoxia Pulmonary Hypertension,HPH)是高原心脏病发病的中心环节,以持续性的肺血管的收缩和肺血管改建为基本特征。HPH会导致右心负荷加重,进而引起右心功能不全,严重者可发展为右心衰竭甚至死亡。HPH的发生机制较为复杂,目前仍不十分清楚,近年来研究发现,肺血管炎症免疫反应和肺血管活性物质分泌失衡是HPH发生过程中的重要机制,而细胞黏附分子(cell adhesion molecules,CAMs)和内皮素1(Endothelin 1,ET-1)表达增多是介导肺血管炎症反应和血管张力增加的两个关键因素。我们前期研究发现,低氧可诱导染色质重构蛋白Brm(brahma)表达上调。Brm在CAMs和ET-1的转录调控中发挥表观遗传调控作用,并在HPH肺血管炎症反应、肺血管结构改建及右心室肥厚中发挥重要作用。但低氧如何诱导Brm表达增多,其机制目前尚不明确。Brm是染色质重构复合物SWI/SNF(switching/sucrose non-fermentation)的核心ATP酶亚单位,也是调节核小体结构改变和基因转录的核心蛋白。Brm可定位到核小体,水解ATP释放能量,实现对核小体的重排和置换,从而在真核基因的转录调控中发挥关键作用。在低氧应激条件下,很大一部分基因的表达受到低氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的转录调控,此外,表观遗传调控机制也在低氧应答反应中发挥着重要作用。其中,以H3K4甲基转移酶复合物(COPASS)介导H3K4甲基化为代表的组蛋白修饰在基因的转录激活中具有重要作用。因此,本研究着重关注了低氧诱导内皮细胞中Brm表达上调的调控机制,结合生物信息学分析,进一步明确低氧诱导因子(HIF-1α)及组蛋白H3K4甲基化修饰在低氧诱导Brm转录激活中的作用及机制。方法:1.以人脐静脉内皮细胞(human Umbilical Vein Endothelial Cells,HUVEC)作为细胞模型,将细胞置于低氧工作站内低氧培养(1%O2、5%CO2、94%N2)一定时间(12h、24h、48h)后,提取mRNA和蛋白质,采用RT-PCR和WB方法检测Brm基因的mRNA及蛋白表达;构建Brm基因的启动子质粒,转染启动子质粒后进行低氧培养,采用双荧光素酶报告基因系统检测Brm启动子活性。2.通过转染外源性的HIF-1α过表达质粒或siRNA干扰内皮细胞中内源性的HIF-1α表达后,采用双荧光素酶报告基因系统检测Brm的启动子活性,采用RT-PCR方法检测Brm的mRNA表达,采用WB方法检测Brm的蛋白表达。并进一步采用染色质免疫共沉淀(Chromatin Immunoprecipitation,ChIP)技术检测低氧条件下内皮细胞中HIF-1α与Brm启动子的结合变化,明确HIF-1α活化Brm转录的机制3.通过向内皮细胞中转染外源性的H3K4甲基化转移酶复合物核心亚单位Wdr5(WD Repeat Domain 5)和Ash2l(ASH2 Like Histone Lysine Methyltransferase Complex Subunit)过表达质粒或siRNA干扰内源性的Wdr5、Ash2l表达后,采用双荧光素酶报告基因系统检测Brm的启动子活性,采用RT-PCR方法检测Brm的mRNA表达,采用WB方法检测Brm的蛋白表达。采用ChIP技术检测低氧处理的内皮细胞中Brm启动子区域H3K4甲基化水平变化情况。结果:1.内皮细胞中转染Brm启动子质粒后低氧(1%O2)培养24h,用双荧光素酶报告基因检测系统检测Brm启动子活性。结果显示:与常氧组比,低氧可显著升高Brm启动子活性;低氧培养内皮细胞不同时间(12h、24h、48h)后与常氧对照组相比Brm的mRNA和蛋白表达水平显著上调,并随低氧培养时间延长逐渐增加,有显著的时效关系。2.与常氧对照组相比,低氧刺激可显著增加内皮细胞中HIF-1α与Brm启动子区域的结合;向内皮细胞中成功导入外源性的HIF-1α过表达质粒,显著提高HIF-1α的mRNA及蛋白表达水平。内皮细胞中过表达HIF-1α能显著增强低氧诱导的Brm的启动子活性,Brm的mRNA及蛋白表达也显著增加;采用siRNA干扰成功内皮细胞中内源性的HIF-1α表达后,HIF-1α的mRNA及蛋白表达水平显著受到抑制。干扰内皮细胞中HIF-1α表达能显著抑制低氧诱导的Brm的启动子活性,Brm的mRNA及蛋白表达也显著降低。3.与常氧对照组相比,低氧处理组Brm启动子周围的H3K4的三甲基化水平显著增加;内皮细胞中成功导入外源性的Wdr5和Ash2l过表达质粒可显著上调Wdr5和Ash2l的mRNA及蛋白表达,低氧时Brm的启动子活性、mRNA及蛋白表达水平较常氧对照组显著升高;采用siRNA干扰内皮细胞中内源性的Wdr5和Ash2l表达后,低氧时Brm的启动子活性、mRNA及蛋白表达水平较常氧对照组显著降低。结论:1.低氧可显著增加内皮细胞中Brm的启动子活性,上调其mRNA和蛋白表达水平,表明转录激活是低氧上调内皮细胞Brm表达的重要机制。2.低氧可促进HIF-1α与Brm启动子结合,并调控Brm的转录和表达,表明HIF-1α在低氧诱导内皮细胞中Brm表达上调中发挥重要的转录调控作用。3.内皮细胞中Brm的转录活性同时受其启动子周围H3K4三甲基化水平的影响,低氧可显著增加Brm启动子区域H3K4的三甲基化水平,进而调节Brm的转录和表达,可能是低氧调控Brm转录、表达的又一重要机制。低氧可能促进HIF-1α与组蛋白甲基化转移酶相互作用,共同调控Brm转录、表达,这有待进一步实验验证。
[Abstract]:Background: hypoxic pulmonary hypertension (Hypoxia Pulmonary Hypertension, HPH) is the central link in the pathogenesis of high altitude heart disease. Persistent pulmonary vascular contraction and pulmonary vascular remodeling are the basic features of.HPH, which can lead to the aggravation of the right heart load and cause the right heart dysfunction, and the serious person can develop to right heart failure or even death.HPH. The system of vitality is relatively complex and is still not very clear. In recent years, it has been found that the immune response to pulmonary vascular inflammation and the imbalance of the secretion of vasoactive substances in the lungs are important mechanisms in the process of HPH, and the increase in the expression of cell adhesion molecules (cell adhesion molecules, CAMs) and endothelin 1 (Endothelin 1, ET-1) is mediated by pulmonary vascular inflammatory reaction and The two key factors of vascular tension increase. Our previous study found that hypoxia can induce the expression of chromatin remodeling protein Brm (Brahma) expression to play epigenetic regulation in the transcription regulation of CAMs and ET-1, and play an important role in HPH pulmonary vascular inflammation, pulmonary vascular remodeling and right ventricular hypertrophy. It is not clear that.Brm is the core ATP subunit of the chromatin restructure complex SWI/SNF (switching/sucrose non-fermentation), and the core protein.Brm regulating the structural change of the nucleosome and gene transcription,.Brm can be located to the nucleosome, the release of energy from ATP, and the rearrangement and replacement of the nucleosome by the hydrolysis of the nucleosome structure and gene transcription, the mechanism is not yet clear. It plays a key role in the transcriptional regulation of eukaryotic genes. Under the condition of hypoxia stress, the expression of a large number of genes is regulated by the transcription of hypoxia inducible factor-1 alpha (HIF-1 a). In addition, epigenetic regulation also plays an important role in the response to hypoxia response. Among them, H3K4 methyltransferase is used. Complex (COPASS) mediated H3K4 methylation as the representative of histone modification plays an important role in gene transcription activation. Therefore, this study focuses on the regulation mechanism of up regulation of Brm expression in hypoxia induced endothelial cells. Combined with bioinformatics analysis, the hypoxic inducible factor (HIF-1 alpha) and histone H3K4 methylation modification are further clarified. The role and mechanism in the activation of Brm transcriptional activation in hypoxia. Methods: 1. human Umbilical Vein Endothelial Cells (HUVEC) was used as a cell model, and cells were placed in low oxygen station (1%O2,5%CO2,94%N2) for a certain time (12h, 24h, 48h) to extract mRNA and protein. The mRNA and protein expression of the M gene; construction of the promoter plasmid of the Brm gene, transfection of the promoter plasmid and hypoxic culture. The double luciferase reporter gene system was used to detect the Brm promoter activity.2. by transfection of exogenous HIF-1 alpha overexpressed plasmid or siRNA to interfere with endogenous HIF-1 alpha expression in endothelial cells, and double luciferase was used. The promoter activity of Brm was detected by the reporter gene system. The expression of mRNA in Brm was detected by RT-PCR method. The protein expression of Brm was detected by WB method. The combination of Chromatin Immunoprecipitation, ChIP (Chromatin Immunoprecipitation, ChIP) was used to detect the binding changes of HIF-1 A and Brm promoter in endothelial cells under hypoxic conditions, and the HIF-1 alpha was clearly defined. The mechanism for activating Brm transcription 3. through transfection of exogenous H3K4 methylation transferase complex core subunit Wdr5 (WD Repeat Domain 5) and Ash2l (ASH2 Like Histone Lysine Methyltransferase) over expressed plasmids or interfering endogenous plasmids, using double Luciferase Report The mRNA expression of Brm was detected by RT-PCR method and WB method was used to detect the protein expression of Brm. The H3K4 methylation level of Brm promoter region in hypoxia treated endothelial cells was detected by ChIP technique. Results: 1. endothelial cells transfected with Brm promoter plasmid after plasmid hypoxia (1%O2) culture 24h, using double The Brm promoter activity was detected by the luciferase reporter gene detection system. The results showed that compared with the normal oxygen group, the hypoxia could significantly increase the activity of the Brm promoter, and the mRNA and protein expression levels of Brm in the hypoxia cultured endothelial cells were up to rise after different time (12h, 24h, 48h), and increased gradually with the prolongation of the hypoxia culture time. Compared with the normoxic control group.2., hypoxia stimulation significantly increased the binding of HIF-1 alpha in the endothelial cells to the Brm promoter region, and successfully introduced the exogenous HIF-1 alpha overexpressed plasmid to the endothelial cells, which significantly enhanced the mRNA and protein expression level of HIF-1 alpha. The overexpression of HIF-1 a in endothelial cells could significantly enhance the hypoxia induction. The promoter activity of Brm and the expression of mRNA and protein in Brm also increased significantly. The expression of mRNA and protein in HIF-1 alpha was significantly inhibited after siRNA interference in the endogenous HIF-1 alpha expression in endothelial cells. The interference of HIF-1 a expression in endothelial cells could significantly inhibit the promoter activity of the Brm induced Brm, mRNA and protein expression of Brm. Also significantly decreased the level of trimethylation of H3K4 around the Brm promoter in the hypoxia treatment group compared with the normal oxygen control group, and the successful introduction of exogenous Wdr5 and Ash2l overexpressed plasmids in the endothelial cells could significantly increase the mRNA and protein expression of Wdr5 and Ash2l, the promoter activity of Brm in hypoxia, and the expression level of mRNA and protein more frequently than that in the hypoxia group. The control group increased significantly; after siRNA interference with endogenous Wdr5 and Ash2l expression in endothelial cells, the promoter activity of Brm and the expression level of mRNA and protein in hypoxia were significantly lower than that of the normal oxygen control group. Conclusion: 1. hypoxia can significantly increase the promoter activity of Brm in endothelial cells, and regulate its mRNA and protein expression level, indicating that the transcriptional activation is The important mechanism of hypoxia up regulation of the expression of Brm in endothelial cells.2. hypoxia can promote the binding of HIF-1 alpha to Brm promoter and regulate the transcription and expression of Brm, which indicates that HIF-1 alpha plays an important transcriptional regulation in the up regulation of Brm expression in hypoxia induced endothelial cells, and the transcriptional activity of Brm in.3. endothelial cells is simultaneously affected by H3K4 trimethylation of the promoter around its promoter. Hypoxia can significantly increase the level of trimethylation of H3K4 in the promoter region of Brm, and then regulate the transcription and expression of Brm. It may be another important mechanism for the regulation of Brm transcription and expression in hypoxia. Hypoxia may promote the interaction of HIF-1 alpha with histone methyltransferase and regulate the transcription and expression of Brm, which need to be further tested. Certificate.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R594.3

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