内质网应激调控炎症反应在高血压心脏损伤中的分子机制研究

发布时间：2018-05-12 08:52

本文选题：高血压心脏损伤 + 炎症反应　；参考：《首都医科大学》2016年博士论文

【摘要】：背景高血压的危害性在于它会导致靶器官损伤,其中心脏损伤非常重要,在此过程中炎症反应发挥十分重要的作用。我们研究已经证明,骨髓来源的炎症细胞,包括巨噬细胞,T细胞和中性粒细胞,在高血压心脏损伤中发挥重要作用,但其中的分子机制尚未在整体水平上进行阐明。全基因组学(RNA-Seq)技术为基础的转录组研究,能够从整体水平研究基因功能以及基因结构,揭示特定生物学过程以及疾病发生过程中的分子机理,但是目前RNA-Seq技术尚未运用于高血压诱导的心脏损伤研究中。大量研究表明内质网应激(ERS)信号通路的激活可以调控炎症反应的发生发展,并参与到多种心血管疾病中。然而,ERS是否参与高血压诱导的心脏炎症反应,以及ERS在此过程中发挥着什么样的作用仍不清楚。所以本研究使用RNA-Seq技术在整体基因组水平研究了炎症反应在高血压心脏损伤的分子机制,以及高血压早期ERS在高血压诱导的心脏炎症损伤中的作用。目的Ang II灌注复制小鼠高血压模型于1、3、7天收取心脏组织提取RNA,采用RNA-Seq进行动态筛选,采用生物学信息分析软件进行在线分析,寻找早期升高最明显的信号通路及ERS相关的信号通路,进而采用ERS信号通路相关分子的敲除小鼠复制高血压模型,观察心脏组织中炎症细胞、炎症因子变化。在整体基因组水平明确炎症反应在高血压心脏损伤的分子机制;并明确ERS在高血压导致心脏损伤过程中的作用,阐明ERS参与心脏损伤过程中的分子机理。方法1.高血压心脏损伤模型制备:采用持续皮下微量泵灌注angii的方法建立高血压心脏损伤模型,灌注angii的剂量为1500ng/kg/min,灌注的时间分别为1、3、7天,设置微量泵灌注生理盐水为对照组(sham)。angii灌注后监测血压高于140/90mmhg提示模型建立成功。2.血压监测:采用尾动脉套管法血压测量仪在angii灌注前测量小鼠基础血压;并在angii灌注后连续测定小鼠血压,明确高血压模型是否建立成功。3.心功能测定:使用visualsonicsvevo2100小动物超声在小鼠angii灌注前测定小鼠基础心功能,并在angii灌注后第7天测量高血压小鼠的心脏功能,明确心功能相关指标的改变。4.炎症细胞浸润检测:采用流式细胞术测定angii灌注前和angii灌注后心脏组织中浸润炎症细胞的种类和数量的变化,包括中性粒细胞、巨噬细胞、t细胞等。5.心脏纤维化检测:采用masson三色染色法,天狼猩红染色法,免疫组化染色法(α-sma、tgf-β1)以及realtime-pcr(collagen1,collagen3和fibronectin)等方法检测angii灌注前和angii灌注后心脏组织中纤维化形成情况。6.基因组变化的检测:运用rna-seq技术(illuminahiseqtm2000)对wt小鼠angii灌注后0、1、3、7天的rna样品进行测序,并对差异表达基因进行表达模式聚类分析、go功能显著性富集分析、pathway显著性富集分析和蛋白质相互作用网络分析。7.心脏炎症因子的检测:使用rna-seq技术检测和分析炎症因子相关基因的改变,并采用realtime-pcr对相应炎症因子的mrna表达水平进行验证(s100a8、s100a9、cxcl1、cxcl2、ccl2、ccl9等)。8.骨髓移植:8-12周龄的wt和chop-/-受体小鼠钴60放射源10gy进行照射后,4-8小时内回输供体小鼠骨髓细胞5x106个/只,放入干净的spf环境下进行饲养8周进行骨髓重建。在此期间饮水为加广谱抗真菌和抗细菌抗生素、ph=2.0的无菌水,8周后改为正常饮水,并可进行相应的实验。9.小鼠骨髓中性粒细胞:采用密度梯度离心法分离骨髓中性粒细胞。10.凋亡及凋亡细胞类型:tunel染色法检测心脏组织中凋亡的细胞,并使用免疫荧光共染的方法确定凋亡细胞的类型。11.凋亡信号通路检测:westernblot检测凋亡相关蛋白bcl-2、bcl-xl等的改变。12.统计学分析:所有数据都以平均值±标准差(x±sem)表示。符合正态分布的计量资料,两组间比较采用成组t检验,不符合正态分布的计量资料采用秩和检验。p0.05为差异有统计学意义。所有数据采用graphpadprism软件进行统计分析。结果第一部分1.angii诱导高血压心脏炎症和纤维化损伤的模型建立成功。2.差异表达基因(degs)分析显示,与对照组sham相比,angii灌注1天、3天、7天后发生变化的基因共有1801个。3.degs的go分析显示angii灌注后,富集的go通路包括对内源性刺激的反应,对急性应激的反应,免疫系统的反应,细胞外组织结构的重建以及细胞外基质的重建等。4.degs的kegg信号通路分析显示angii灌注后,细胞因子-细胞因子相互作用通路,趋化因子信号通路,细胞外基质(ecm)受体相互作用,细胞粘附以及tgf-β等信号通路被激活。5.degs的聚类分析显示angii灌注后心脏中的差异表达1801个基因可被划分为16种表达模式,每一种代表该组基因在angii灌注后具有相同的表达模式。6.degs的蛋白-蛋白相互作用网络分析显示ubiquitinc与周围118个基因相互作用;rnf2,eed,npm1,myc等可与其他10个以上的基因相互作用。第二部分7.rna-seq和rt-pcr显示angii灌注后引起内质网应激信号通路的激活。8.内质网应激信号通路中的一个关键分子为chop,chop缺失后增加angii诱导的心脏中炎症因子的表达。9.流式细胞术检测显示chop缺失后增加angii诱导的心脏中巨噬细胞、中性粒细胞、t细胞等炎症细胞的浸润。10.masson染色和rt-pcr显示chop缺失后加重angii诱导的心脏纤维化损伤。11.骨髓移植实验显示骨髓来源细胞表达的chop,在angii诱导的高血压心脏损伤中发挥重要作用。12.chop缺失减少angii灌注后心脏组织中中性粒细胞的凋亡。13.chop缺失减少体外中性粒细胞凋亡。结论第一部分1.与sham相比,angii灌注心脏1天、3天、7天后,诱导心脏组织中1,801个基因差异表达。2.对内源性刺激的反应,对急性应激的反应,免疫系统的反应,细胞外组织结构的重建以及细胞外基质的重建等go信号通路在angii诱导的心脏损伤中发挥重要作用。3.细胞因子-细胞因子相互作用通路,趋化因子信号通路,细胞外基质(ecm)受体相互作用,细胞粘附以及tgf-β等信号通路在angii诱导的心脏损伤中发挥重要作用。4.angii灌注后心脏中的差异表达的1801个基因可被划分为16种表达模式,并在高血压心脏损伤的过程中发挥重要作用。5.ubiquitinc,rnf2,eed,npm1以及myc等基因通过与周围其他多种基因相互作用在高血压心脏损伤的过程中发挥重要作用。第二部分6.Ang II灌注早期可诱导内质网应激发生。7.CHOP缺失后增加Ang II诱导的心脏炎症因子表达、炎症细胞浸润,并最终加重高血压心脏纤维化损伤。8.骨髓来源细胞表达的CHOP在Ang II诱导的高血压心脏损伤中发挥重要作用。9.早期内质网应激和CHOP在高血压心脏损伤中发挥保护作用,可能是通过减少中性粒细胞的凋亡,延迟早期炎症清除,从而加重心脏炎症反应和心脏损伤。综上所述,在Ang II诱导的高血压心脏损伤过程中,炎症反应发挥着重要作用。机体会启动关于急性应激反应,免疫系统反应,细胞外组织结构重建以及细胞外基质重建等信号通路参与到该损伤过程中。其中Ang II灌注后的高血压早期会启动内质网应激信号通路,当内质网应激异常(CHOP缺失)时则会加重高血压诱导的心脏炎症和纤维化损伤。
[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.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.3

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