本文選題：低氧性肺動(dòng)脈高壓 + Brm　； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景:低氧性肺動(dòng)脈高壓(Hypoxia Pulmonary Hypertension,HPH)是高原心臟病發(fā)病的中心環(huán)節(jié),以持續(xù)性的肺血管的收縮和肺血管改建為基本特征。HPH會(huì)導(dǎo)致右心負(fù)荷加重,進(jìn)而引起右心功能不全,嚴(yán)重者可發(fā)展為右心衰竭甚至死亡。HPH的發(fā)生機(jī)制較為復(fù)雜,目前仍不十分清楚,近年來(lái)研究發(fā)現(xiàn),肺血管炎癥免疫反應(yīng)和肺血管活性物質(zhì)分泌失衡是HPH發(fā)生過(guò)程中的重要機(jī)制,而細(xì)胞黏附分子(cell adhesion molecules,CAMs)和內(nèi)皮素1(Endothelin 1,ET-1)表達(dá)增多是介導(dǎo)肺血管炎癥反應(yīng)和血管張力增加的兩個(gè)關(guān)鍵因素。我們前期研究發(fā)現(xiàn),低氧可誘導(dǎo)染色質(zhì)重構(gòu)蛋白Brm(brahma)表達(dá)上調(diào)。Brm在CAMs和ET-1的轉(zhuǎn)錄調(diào)控中發(fā)揮表觀遺傳調(diào)控作用,并在HPH肺血管炎癥反應(yīng)、肺血管結(jié)構(gòu)改建及右心室肥厚中發(fā)揮重要作用。但低氧如何誘導(dǎo)Brm表達(dá)增多,其機(jī)制目前尚不明確。Brm是染色質(zhì)重構(gòu)復(fù)合物SWI/SNF(switching/sucrose non-fermentation)的核心ATP酶亞單位,也是調(diào)節(jié)核小體結(jié)構(gòu)改變和基因轉(zhuǎn)錄的核心蛋白。Brm可定位到核小體,水解ATP釋放能量,實(shí)現(xiàn)對(duì)核小體的重排和置換,從而在真核基因的轉(zhuǎn)錄調(diào)控中發(fā)揮關(guān)鍵作用。在低氧應(yīng)激條件下,很大一部分基因的表達(dá)受到低氧誘導(dǎo)因子(hypoxia inducible factor-1α,HIF-1α)的轉(zhuǎn)錄調(diào)控,此外,表觀遺傳調(diào)控機(jī)制也在低氧應(yīng)答反應(yīng)中發(fā)揮著重要作用。其中,以H3K4甲基轉(zhuǎn)移酶復(fù)合物(COPASS)介導(dǎo)H3K4甲基化為代表的組蛋白修飾在基因的轉(zhuǎn)錄激活中具有重要作用。因此,本研究著重關(guān)注了低氧誘導(dǎo)內(nèi)皮細(xì)胞中Brm表達(dá)上調(diào)的調(diào)控機(jī)制,結(jié)合生物信息學(xué)分析,進(jìn)一步明確低氧誘導(dǎo)因子(HIF-1α)及組蛋白H3K4甲基化修飾在低氧誘導(dǎo)Brm轉(zhuǎn)錄激活中的作用及機(jī)制。方法:1.以人臍靜脈內(nèi)皮細(xì)胞(human Umbilical Vein Endothelial Cells,HUVEC)作為細(xì)胞模型,將細(xì)胞置于低氧工作站內(nèi)低氧培養(yǎng)(1%O2、5%CO2、94%N2)一定時(shí)間(12h、24h、48h)后,提取mRNA和蛋白質(zhì),采用RT-PCR和WB方法檢測(cè)Brm基因的mRNA及蛋白表達(dá);構(gòu)建Brm基因的啟動(dòng)子質(zhì)粒,轉(zhuǎn)染啟動(dòng)子質(zhì)粒后進(jìn)行低氧培養(yǎng),采用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)Brm啟動(dòng)子活性。2.通過(guò)轉(zhuǎn)染外源性的HIF-1α過(guò)表達(dá)質(zhì)�；騭iRNA干擾內(nèi)皮細(xì)胞中內(nèi)源性的HIF-1α表達(dá)后,采用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)Brm的啟動(dòng)子活性,采用RT-PCR方法檢測(cè)Brm的mRNA表達(dá),采用WB方法檢測(cè)Brm的蛋白表達(dá)。并進(jìn)一步采用染色質(zhì)免疫共沉淀(Chromatin Immunoprecipitation,ChIP)技術(shù)檢測(cè)低氧條件下內(nèi)皮細(xì)胞中HIF-1α與Brm啟動(dòng)子的結(jié)合變化,明確HIF-1α活化Brm轉(zhuǎn)錄的機(jī)制3.通過(guò)向內(nèi)皮細(xì)胞中轉(zhuǎn)染外源性的H3K4甲基化轉(zhuǎn)移酶復(fù)合物核心亞單位Wdr5(WD Repeat Domain 5)和Ash2l(ASH2 Like Histone Lysine Methyltransferase Complex Subunit)過(guò)表達(dá)質(zhì)�；騭iRNA干擾內(nèi)源性的Wdr5、Ash2l表達(dá)后,采用雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)Brm的啟動(dòng)子活性,采用RT-PCR方法檢測(cè)Brm的mRNA表達(dá),采用WB方法檢測(cè)Brm的蛋白表達(dá)。采用ChIP技術(shù)檢測(cè)低氧處理的內(nèi)皮細(xì)胞中Brm啟動(dòng)子區(qū)域H3K4甲基化水平變化情況。結(jié)果:1.內(nèi)皮細(xì)胞中轉(zhuǎn)染Brm啟動(dòng)子質(zhì)粒后低氧(1%O2)培養(yǎng)24h,用雙熒光素酶報(bào)告基因檢測(cè)系統(tǒng)檢測(cè)Brm啟動(dòng)子活性。結(jié)果顯示:與常氧組比,低氧可顯著升高Brm啟動(dòng)子活性;低氧培養(yǎng)內(nèi)皮細(xì)胞不同時(shí)間(12h、24h、48h)后與常氧對(duì)照組相比Brm的mRNA和蛋白表達(dá)水平顯著上調(diào),并隨低氧培養(yǎng)時(shí)間延長(zhǎng)逐漸增加,有顯著的時(shí)效關(guān)系。2.與常氧對(duì)照組相比,低氧刺激可顯著增加內(nèi)皮細(xì)胞中HIF-1α與Brm啟動(dòng)子區(qū)域的結(jié)合;向內(nèi)皮細(xì)胞中成功導(dǎo)入外源性的HIF-1α過(guò)表達(dá)質(zhì)粒,顯著提高HIF-1α的mRNA及蛋白表達(dá)水平。內(nèi)皮細(xì)胞中過(guò)表達(dá)HIF-1α能顯著增強(qiáng)低氧誘導(dǎo)的Brm的啟動(dòng)子活性,Brm的mRNA及蛋白表達(dá)也顯著增加;采用siRNA干擾成功內(nèi)皮細(xì)胞中內(nèi)源性的HIF-1α表達(dá)后,HIF-1α的mRNA及蛋白表達(dá)水平顯著受到抑制。干擾內(nèi)皮細(xì)胞中HIF-1α表達(dá)能顯著抑制低氧誘導(dǎo)的Brm的啟動(dòng)子活性,Brm的mRNA及蛋白表達(dá)也顯著降低。3.與常氧對(duì)照組相比,低氧處理組Brm啟動(dòng)子周圍的H3K4的三甲基化水平顯著增加;內(nèi)皮細(xì)胞中成功導(dǎo)入外源性的Wdr5和Ash2l過(guò)表達(dá)質(zhì)粒可顯著上調(diào)Wdr5和Ash2l的mRNA及蛋白表達(dá),低氧時(shí)Brm的啟動(dòng)子活性、mRNA及蛋白表達(dá)水平較常氧對(duì)照組顯著升高;采用siRNA干擾內(nèi)皮細(xì)胞中內(nèi)源性的Wdr5和Ash2l表達(dá)后,低氧時(shí)Brm的啟動(dòng)子活性、mRNA及蛋白表達(dá)水平較常氧對(duì)照組顯著降低。結(jié)論:1.低氧可顯著增加內(nèi)皮細(xì)胞中Brm的啟動(dòng)子活性,上調(diào)其mRNA和蛋白表達(dá)水平,表明轉(zhuǎn)錄激活是低氧上調(diào)內(nèi)皮細(xì)胞Brm表達(dá)的重要機(jī)制。2.低氧可促進(jìn)HIF-1α與Brm啟動(dòng)子結(jié)合,并調(diào)控Brm的轉(zhuǎn)錄和表達(dá),表明HIF-1α在低氧誘導(dǎo)內(nèi)皮細(xì)胞中Brm表達(dá)上調(diào)中發(fā)揮重要的轉(zhuǎn)錄調(diào)控作用。3.內(nèi)皮細(xì)胞中Brm的轉(zhuǎn)錄活性同時(shí)受其啟動(dòng)子周圍H3K4三甲基化水平的影響,低氧可顯著增加Brm啟動(dòng)子區(qū)域H3K4的三甲基化水平,進(jìn)而調(diào)節(jié)Brm的轉(zhuǎn)錄和表達(dá),可能是低氧調(diào)控Brm轉(zhuǎn)錄、表達(dá)的又一重要機(jī)制。低氧可能促進(jìn)HIF-1α與組蛋白甲基化轉(zhuǎn)移酶相互作用,共同調(diào)控Brm轉(zhuǎn)錄、表達(dá),這有待進(jìn)一步實(shí)驗(yàn)驗(yàn)證。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R594.3

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相關(guān)期刊論文 前1條

1 高文祥;高鈺琪;;慢性高原病分型、診斷與治療的研究進(jìn)展[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2016年05期

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本文編號(hào)：1882124