本文選題：高血壓心臟損傷 + 炎癥反應(yīng)�。� 參考：《首都醫(yī)科大學》2016年博士論文

【摘要】：背景高血壓的危害性在于它會導致靶器官損傷,其中心臟損傷非常重要,在此過程中炎癥反應(yīng)發(fā)揮十分重要的作用。我們研究已經(jīng)證明,骨髓來源的炎癥細胞,包括巨噬細胞,T細胞和中性粒細胞,在高血壓心臟損傷中發(fā)揮重要作用,但其中的分子機制尚未在整體水平上進行闡明。全基因組學(RNA-Seq)技術(shù)為基礎(chǔ)的轉(zhuǎn)錄組研究,能夠從整體水平研究基因功能以及基因結(jié)構(gòu),揭示特定生物學過程以及疾病發(fā)生過程中的分子機理,但是目前RNA-Seq技術(shù)尚未運用于高血壓誘導的心臟損傷研究中。大量研究表明內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)信號通路的激活可以調(diào)控炎癥反應(yīng)的發(fā)生發(fā)展,并參與到多種心血管疾病中。然而,ERS是否參與高血壓誘導的心臟炎癥反應(yīng),以及ERS在此過程中發(fā)揮著什么樣的作用仍不清楚。所以本研究使用RNA-Seq技術(shù)在整體基因組水平研究了炎癥反應(yīng)在高血壓心臟損傷的分子機制,以及高血壓早期ERS在高血壓誘導的心臟炎癥損傷中的作用。目的Ang II灌注復(fù)制小鼠高血壓模型于1、3、7天收取心臟組織提取RNA,采用RNA-Seq進行動態(tài)篩選,采用生物學信息分析軟件進行在線分析,尋找早期升高最明顯的信號通路及ERS相關(guān)的信號通路,進而采用ERS信號通路相關(guān)分子的敲除小鼠復(fù)制高血壓模型,觀察心臟組織中炎癥細胞、炎癥因子變化。在整體基因組水平明確炎癥反應(yīng)在高血壓心臟損傷的分子機制;并明確ERS在高血壓導致心臟損傷過程中的作用,闡明ERS參與心臟損傷過程中的分子機理。方法1.高血壓心臟損傷模型制備:采用持續(xù)皮下微量泵灌注angii的方法建立高血壓心臟損傷模型,灌注angii的劑量為1500ng/kg/min,灌注的時間分別為1、3、7天,設(shè)置微量泵灌注生理鹽水為對照組(sham)。angii灌注后監(jiān)測血壓高于140/90mmhg提示模型建立成功。2.血壓監(jiān)測:采用尾動脈套管法血壓測量儀在angii灌注前測量小鼠基礎(chǔ)血壓;并在angii灌注后連續(xù)測定小鼠血壓,明確高血壓模型是否建立成功。3.心功能測定:使用visualsonicsvevo2100小動物超聲在小鼠angii灌注前測定小鼠基礎(chǔ)心功能,并在angii灌注后第7天測量高血壓小鼠的心臟功能,明確心功能相關(guān)指標的改變。4.炎癥細胞浸潤檢測:采用流式細胞術(shù)測定angii灌注前和angii灌注后心臟組織中浸潤炎癥細胞的種類和數(shù)量的變化,包括中性粒細胞、巨噬細胞、t細胞等。5.心臟纖維化檢測:采用masson三色染色法,天狼猩紅染色法,免疫組化染色法(α-sma、tgf-β1)以及realtime-pcr(collagen1,collagen3和fibronectin)等方法檢測angii灌注前和angii灌注后心臟組織中纖維化形成情況。6.基因組變化的檢測:運用rna-seq技術(shù)(illuminahiseqtm2000)對wt小鼠angii灌注后0、1、3、7天的rna樣品進行測序,并對差異表達基因進行表達模式聚類分析、go功能顯著性富集分析、pathway顯著性富集分析和蛋白質(zhì)相互作用網(wǎng)絡(luò)分析。7.心臟炎癥因子的檢測:使用rna-seq技術(shù)檢測和分析炎癥因子相關(guān)基因的改變,并采用realtime-pcr對相應(yīng)炎癥因子的mrna表達水平進行驗證(s100a8、s100a9、cxcl1、cxcl2、ccl2、ccl9等)。8.骨髓移植:8-12周齡的wt和chop-/-受體小鼠鈷60放射源10gy進行照射后,4-8小時內(nèi)回輸供體小鼠骨髓細胞5x106個/只,放入干凈的spf環(huán)境下進行飼養(yǎng)8周進行骨髓重建。在此期間飲水為加廣譜抗真菌和抗細菌抗生素、ph=2.0的無菌水,8周后改為正常飲水,并可進行相應(yīng)的實驗。9.小鼠骨髓中性粒細胞:采用密度梯度離心法分離骨髓中性粒細胞。10.凋亡及凋亡細胞類型:tunel染色法檢測心臟組織中凋亡的細胞,并使用免疫熒光共染的方法確定凋亡細胞的類型。11.凋亡信號通路檢測:westernblot檢測凋亡相關(guān)蛋白bcl-2、bcl-xl等的改變。12.統(tǒng)計學分析:所有數(shù)據(jù)都以平均值±標準差(x±sem)表示。符合正態(tài)分布的計量資料,兩組間比較采用成組t檢驗,不符合正態(tài)分布的計量資料采用秩和檢驗。p0.05為差異有統(tǒng)計學意義。所有數(shù)據(jù)采用graphpadprism軟件進行統(tǒng)計分析。結(jié)果第一部分1.angii誘導高血壓心臟炎癥和纖維化損傷的模型建立成功。2.差異表達基因(degs)分析顯示,與對照組sham相比,angii灌注1天、3天、7天后發(fā)生變化的基因共有1801個。3.degs的go分析顯示angii灌注后,富集的go通路包括對內(nèi)源性刺激的反應(yīng),對急性應(yīng)激的反應(yīng),免疫系統(tǒng)的反應(yīng),細胞外組織結(jié)構(gòu)的重建以及細胞外基質(zhì)的重建等。4.degs的kegg信號通路分析顯示angii灌注后,細胞因子-細胞因子相互作用通路,趨化因子信號通路,細胞外基質(zhì)(ecm)受體相互作用,細胞粘附以及tgf-β等信號通路被激活。5.degs的聚類分析顯示angii灌注后心臟中的差異表達1801個基因可被劃分為16種表達模式,每一種代表該組基因在angii灌注后具有相同的表達模式。6.degs的蛋白-蛋白相互作用網(wǎng)絡(luò)分析顯示ubiquitinc與周圍118個基因相互作用;rnf2,eed,npm1,myc等可與其他10個以上的基因相互作用。第二部分7.rna-seq和rt-pcr顯示angii灌注后引起內(nèi)質(zhì)網(wǎng)應(yīng)激信號通路的激活。8.內(nèi)質(zhì)網(wǎng)應(yīng)激信號通路中的一個關(guān)鍵分子為chop,chop缺失后增加angii誘導的心臟中炎癥因子的表達。9.流式細胞術(shù)檢測顯示chop缺失后增加angii誘導的心臟中巨噬細胞、中性粒細胞、t細胞等炎癥細胞的浸潤。10.masson染色和rt-pcr顯示chop缺失后加重angii誘導的心臟纖維化損傷。11.骨髓移植實驗顯示骨髓來源細胞表達的chop,在angii誘導的高血壓心臟損傷中發(fā)揮重要作用。12.chop缺失減少angii灌注后心臟組織中中性粒細胞的凋亡。13.chop缺失減少體外中性粒細胞凋亡。結(jié)論第一部分1.與sham相比,angii灌注心臟1天、3天、7天后,誘導心臟組織中1,801個基因差異表達。2.對內(nèi)源性刺激的反應(yīng),對急性應(yīng)激的反應(yīng),免疫系統(tǒng)的反應(yīng),細胞外組織結(jié)構(gòu)的重建以及細胞外基質(zhì)的重建等go信號通路在angii誘導的心臟損傷中發(fā)揮重要作用。3.細胞因子-細胞因子相互作用通路,趨化因子信號通路,細胞外基質(zhì)(ecm)受體相互作用,細胞粘附以及tgf-β等信號通路在angii誘導的心臟損傷中發(fā)揮重要作用。4.angii灌注后心臟中的差異表達的1801個基因可被劃分為16種表達模式,并在高血壓心臟損傷的過程中發(fā)揮重要作用。5.ubiquitinc,rnf2,eed,npm1以及myc等基因通過與周圍其他多種基因相互作用在高血壓心臟損傷的過程中發(fā)揮重要作用。第二部分6.Ang II灌注早期可誘導內(nèi)質(zhì)網(wǎng)應(yīng)激發(fā)生。7.CHOP缺失后增加Ang II誘導的心臟炎癥因子表達、炎癥細胞浸潤,并最終加重高血壓心臟纖維化損傷。8.骨髓來源細胞表達的CHOP在Ang II誘導的高血壓心臟損傷中發(fā)揮重要作用。9.早期內(nèi)質(zhì)網(wǎng)應(yīng)激和CHOP在高血壓心臟損傷中發(fā)揮保護作用,可能是通過減少中性粒細胞的凋亡,延遲早期炎癥清除,從而加重心臟炎癥反應(yīng)和心臟損傷。綜上所述,在Ang II誘導的高血壓心臟損傷過程中,炎癥反應(yīng)發(fā)揮著重要作用。機體會啟動關(guān)于急性應(yīng)激反應(yīng),免疫系統(tǒng)反應(yīng),細胞外組織結(jié)構(gòu)重建以及細胞外基質(zhì)重建等信號通路參與到該損傷過程中。其中Ang II灌注后的高血壓早期會啟動內(nèi)質(zhì)網(wǎng)應(yīng)激信號通路,當內(nèi)質(zhì)網(wǎng)應(yīng)激異常(CHOP缺失)時則會加重高血壓誘導的心臟炎癥和纖維化損傷。
[Abstract]:Background the harm of hypertension is that it causes damage to the target organ, in which the heart damage is very important and the inflammatory response plays a very important role in this process. Our study has shown that the inflammatory cells derived from bone marrow, including macrophages, T cells and neutrophils, play an important role in the heart damage of hypertension, but they are important The molecular mechanisms in the genome have not yet been clarified at the overall level. RNA-Seq technology based transcriptional studies can study gene function and gene structure from the overall level, reveal specific biological processes and molecular mechanisms in the process of disease, but RNA-Seq technology has not yet been used to induce hypertension. A large number of studies have shown that the activation of endoplasmic reticulum stress (ERS) signaling pathways can regulate the development of inflammatory responses and participate in a variety of cardiovascular diseases. However, it is not clear whether ERS is involved in hypertension induced cardiac inflammation and what role ERS plays in this process. The molecular mechanism of inflammatory response in hypertensive heart damage and the role of ERS in hypertension induced cardiac inflammation were studied at the overall genomic level by RNA-Seq technique. Objective Ang II perfusion and replication of hypertension model in mice was used to collect RNA for cardiac tissue from 1,3,7 days and select RNA-Seq for dynamic screening. The biological information analysis software carries out online analysis to find the most obvious signal pathways and ERS related signaling pathways in the early stage, and then use the knockout mice of ERS signaling molecules to replicate the hypertension model and observe the changes of inflammatory cells and inflammatory factors in the heart tissues. The molecular mechanism of heart damage and the role of ERS in the process of heart damage caused by hypertension and the molecular mechanism of ERS involved in the heart damage. Method 1. model preparation of hypertensive heart damage: a model of hypertensive heart damage was established by continuous subcutaneous micropump perfusion of AngII, and the dose of AngII was 1500ng/kg/mi N, the time of perfusion was 1,3,7 days respectively, and micropump perfusion of saline was set up as the control group (sham). After.Angii perfusion, the blood pressure monitoring was higher than that of 140/90mmhg, and the blood pressure was measured by the tail artery cannula method of blood pressure measuring instrument before AngII perfusion, and the blood pressure of mice was continuously measured after AngII perfusion, and the blood pressure was determined continuously after AngII perfusion. Whether the hypertensive model was established successfully.3. cardiac function test: using visualsonicsvevo2100 small animal ultrasound to measure the basic cardiac function before AngII perfusion in mice, and measure the cardiac function of the hypertensive mice seventh days after AngII perfusion, and make clear the changes of the cardiac function related indexes of the.4. inflammatory cell infiltration test: the flow cytometry was used to measure the cardiac function. Changes in the types and numbers of infiltrating inflammatory cells in the cardiac tissue before and after AngII perfusion were determined, including neutrophils, macrophages, T cells and other.5. cardiac fibrosis tests: Masson tricolor staining, Sirius scarlet staining, immunohistochemical staining (alpha -sma, tgf- beta 1), and realtime-pcr (collagen1, collagen3 and fibron). Ectin) detection of.6. genome changes in cardiac tissue before and after perfusion of AngII after perfusion and AngII perfusion: RNA-seq technique (illuminahiseqtm2000) was used to sequence the RNA samples of 0,1,3,7 days after AngII perfusion in WT mice and to cluster analysis on the expression pattern of differentially expressed genes and significant enrichment of go function. Analysis of pathway significant enrichment analysis and protein interaction network analysis of the detection of.7. cardiac inflammatory factors: using RNA-seq technique to detect and analyze the changes in genes related to inflammatory factors, and to use realtime-pcr to verify the mRNA expression level of the corresponding inflammatory factors (S100A8, S100A9, CXCL1, cxcl2, CCL2, ccl9, etc.).8. bone marrow transplantation: 8-1 2 weeks old WT and chop-/- receptor mice were irradiated with cobalt 60 source 10GY, and the bone marrow cells of the donor mice were reused for 8 weeks for 8 weeks in 4-8 hours, and the bone marrow cells were reared in a clean SPF environment for 8 weeks. During this period, the drinking water was added to the broad-spectrum antifungal and anti bacterial antibiotics, and the ph= 2 was sterile water, and changed to normal drinking water after 8 weeks. .9. mouse bone marrow neutrophils were tested by the method of density gradient centrifugation to isolate.10. apoptosis and apoptotic cells in bone marrow neutrophils: TUNEL staining was used to detect apoptotic cells in cardiac tissue, and the apoptosis signaling pathway of apoptotic cells was determined by immunofluorescence CO staining: Westernblot detection The change of apoptosis related protein Bcl-2, Bcl-xL and so on.12. statistical analysis: all data were expressed with mean standard deviation (x + SEM). The measurement data conforming to normal distribution were compared with group t test, and the measurement data that did not conform to normal distribution used rank sum test and.P0.05 as difference was statistically significant. All data were graphpadp Rism software was used for statistical analysis. Results the first part of 1.angii induced hypertension and cardiac fibrosis damage model established the successful.2. differential expression gene (DEGs) analysis showed that compared with the control group sham, AngII perfusion was 1 days, 3 days, and 7 days after the change of the go analysis showed AngII perfusion, enriched go through. The pathway includes responses to endogenous stimuli. The response to acute stress, the response of the immune system, the reconstruction of the structure of the extracellular tissue, and the reconstruction of the extracellular matrix,.4.degs KEGG signal pathway analysis shows that after AngII perfusion, the cytokine cytokine interaction pathway, chemokine signaling pathway, and the extracellular matrix (ECM) receptor phase Interaction, cell adhesion and tgf- beta signaling pathway activation.5.degs cluster analysis showed that 1801 genes expressed in the heart after AngII perfusion could be divided into 16 expression patterns, each of which represents the same expression pattern.6.degs after AngII perfusion, the protein protein interaction network analysis showed ubiqu Itinc interacts with the surrounding 118 genes; Rnf2, eed, NPM1, Myc, etc. can interact with more than 10 other genes. Second 7.rna-seq and RT-PCR show that a key component in the activation of the endoplasmic reticulum stress signaling pathway after AngII perfusion is a key component of the.8. endoplasmic reticulum stress signal pathway, which increases the AngII induced heart after chop deletion. Expression of medium inflammatory factors.9. flow cytometry showed that after chop deletion, AngII induced macrophages, neutrophils, T cells and other inflammatory cells were infiltrated.10.masson staining and RT-PCR showed chop deletion aggravated AngII induced cardiac fibrosis injury and.11. bone marrow transplantation experiment showed Cho of bone marrow derived cells. P, which plays an important role in AngII induced hypertensive heart damage,.12.chop deletion reduces the apoptosis.13.chop deletion of neutrophils in cardiac tissue after AngII perfusion and reduces the apoptosis of neutrophils in vitro. Conclusion the first part 1. compared with sham, AngII perfusion was 1 days, 3 days, and 7 days after AngII perfusion, inducing 1801 gene differential expression in cardiac tissue. 2. the response to endogenous stimuli, the response to acute stress, the response of the immune system, the reconstruction of the structure of the extracellular tissue, and the reconstruction of the extracellular matrix, play an important role in the heart damage induced by AngII,.3. cytokine cytokine interaction, chemokine signaling pathway, and extracellular matrix (ECM). Body interaction, cell adhesion and tgf- beta signaling pathway play an important role in AngII induced heart damage. The 1801 genes expressed differently in the heart after.4.angii perfusion can be divided into 16 expression patterns, and play important roles in.5.ubiquitinc, Rnf2, eed, NPM1, and myc in the process of hypertensive heart damage. Interaction with a variety of other genes in the surrounding area plays an important role in the process of hypertensive heart damage. Second part of 6.Ang II perfusion can induce endoplasmic reticulum stress to induce.7.CHOP deletion and increase the expression of cardiac inflammatory factors induced by Ang II, infiltration of inflammatory cells, and ultimately aggravate the.8. marrow of hypertensive cardiac fibrosis. CHOP expressed by source cells plays an important role in Ang II induced hypertensive heart damage,.9. early endoplasmic reticulum stress and CHOP play a protective role in hypertensive heart damage, which may be by reducing the apoptosis of neutrophils and delayed early inflammatory clearance, thus adding center of gravity and inflammation and heart damage. To sum up, in Ang The inflammatory response plays an important role in the process of II induced hypertensive heart damage. The signal pathways involved in the acute stress response, the immune system response, the reconstruction of the extracellular structure and the reconstruction of the extracellular matrix are involved in the injury process. In the early stage of hypertension after Ang II perfusion, the endoplasmic reticulum stress letter will be started. Signal pathway, when endoplasmic reticulum stress abnormality (CHOP deletion), aggravates hypertension induced cardiac inflammation and fibrosis damage.

【學位授予單位】：首都醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R541.3

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