【作者】 陆永光；

【导师】 李浪；

【作者基本信息】 广西医科大学 ， 心血管内科， 2011， 博士

【摘要】 动脉粥样硬化(Atherosclerosis,AS)是慢性炎症反应和免疫性疾病,是冠心病的主要病理基础。在AS斑块中可发现大量的T淋巴细胞,其中以CD4+T淋巴细胞为主,CD4+T淋巴细胞亚群的失衡与急性冠脉综合征(Acute coronary syndrome,ACS)发生关系密切。易损斑块中大量活化的T淋巴细胞降低斑块的稳定性,使斑块易于破裂。不稳定型心绞痛(Unstable angina pectoris,UAP)的患者及急性心肌梗死患者,心肌微血管内存在血小板血栓,在心肌微循环水平形成微栓塞,导致心肌微梗死。因此,CD4+T淋巴细胞可能间接的参与了ACS患者心肌损伤的过程。术后心肌损伤仍然是经皮冠状动脉介入治疗(Percutaneous coronary intervention,PCI)常见的并发症,但其机制目前尚不完全清楚。有研究表明,PCI术前大剂量的他汀可以有效的降低炎症因子水平,保护心肌。因此,炎症和免疫反应也是PCI术后心肌损伤的重要原因之一。还有研究表明,PCI对血管壁的损伤可以导致外周血淋巴细胞的显著激活和氧化应激产物标志物的显著增高,外周血激活的CD4+T淋巴细胞表达的CD18分子可促进白细胞招募并粘附于受损的内皮细胞。因此,炎症反应及以CD4+T淋巴细胞介导的免疫反应直接参与了PCI术后心肌损伤的过程。微小核糖核酸(MicroRNA,miRNA)是一类长约18～25个核苷酸的小分子RNA。近年来发现,miRNA也参对与AS发生发展密切相关的炎性细胞如单核/巨噬细胞、淋巴细胞及内皮细胞的调控。虽然目前miRNA在T淋巴细胞调控中的研究取得了重大的进展,但仍属于起步阶段,并且在不同的疾病状态和疾病发展的不同阶段,miRNA表达和作用亦不尽相同,与T淋巴细胞活化和功能调控相关的miRNA在UAP及PCI术后心肌损伤中的生物学功能尚未阐明。本研究拟通过miRNA基因芯片技术筛选出UAP患者外周血CD4+T淋巴细胞异常表达的miRNA,阐明异常表达的miRNA对CD4+T淋巴细胞活化、分化及功能调控的机制。同时探讨miRNA在PCI术后的变化及与心肌损伤相关性。目的:通过检测UAP患者循环血CD4+T淋巴细胞中基因的miRNA表达谱,筛选与正常对照者差异表达的miRNA,寻找对CD4+T淋巴细胞具有调控作用的miRNA,为进一步阐明miRNA在UAP发病机制中的作用提供基础。方法:选取入住我院的典型UAP患者3例,以疑似不典型冠心病症状而冠脉造影正常的住院患者3例作为正常对照组,抽取新鲜外周静脉血20ml。利用密度梯度离心法分离出UAP患者和正常对照者循环血中的单个核细胞(Peripheral blood mononuclear cell,PBMC),免疫磁珠法(Magnetic cell sorting system,MACS)进一步分离出CD4+T淋巴细胞。采用流式细胞术(Flow cytometry,FCM)检测所分离CD4+T淋巴细胞的纯度,台酚蓝检测活细胞数。Trizol一步法提取细胞总RNA,取40μg总RNA,用聚乙二醇(Polyethylene glycol,PEG)方法分离纯化miRNA。用分光光度计测定RNA的浓度,甲醛变性胶电泳质检RNA的质量。采用Affymetrix miRNA基因表达谱芯片进行杂交,检测CD4+T淋巴细胞miRNA的表达谱。用Affymetrix GeneChip Scanner 3000基因芯片扫描仪进行图像扫描,Affymetrix GeneChip Command Console? 1.1图像分析软件对图像进行分析,把图像信号转化为数字信号,然后用SAM软件(版本3.02)处理数据,筛选出UAP患者和正常对照者CD4+T淋巴细胞差异表达的miRNA。采用实时荧光定量聚合酶链式反应(Real-time polymerase chain reaction,real time PCR)对部分差异表达的miRNA进行验证。结果:miRNA基因芯片筛选结果显示,相对于正常对照者,UAP患者外周血CD4+T淋巴细胞中表达显著上调的miRNA有miR-155, miR-21, miR-424和miR-127-3p,显著下调的有miR-30b和miR-181a。real time PCR进一步验证的结果表明,上述差异表达miRNA的变化趋势及变化倍数与miRNA基因芯片筛选结果一致。结论:筛选得到的UAP患者循环血CD4+T淋巴细胞miRNA差异表达谱,可能与参与了UAP的发生发展。目的:通过研究微小核糖核酸-155(miRNA-155)对UAP患者外周血CD4+T淋巴细胞的调控作用,从而揭示miRNA-155在UAP发病中的作用机制。方法:选取入住我院的UAP患者10例,抽取新鲜外周静脉血。采用密度梯度离心法分离出UAP患者PBMC,MACS法进一步分离出CD4+T淋巴细胞,用无血清RPMI1640培养基调整为2×106/ml,随后分为四组:对照组、miRNA-155模拟体组和阻遏物组,以另一孔转染荧光对照的FAM-siRNA作为荧光对照。对照组加入阴性对照的模拟体(终浓度为50nM),miRNA-155模拟体组中加入的为miRNA-155模拟体(终浓度为50nM),阻遏物组加入的miRNA-155模拟体和miR-155阻遏物(终浓度分别为50nM),同时加入脂质体2000,共转染5h。随后加入植物凝血素刺激CD4+T淋巴细胞活化后,收集细胞及上清。流式细胞术检测CD4+T淋巴细胞Th1和Th2亚群数量,提取CD4+T淋巴细胞总RNA和蛋白,real time PCR检测γ干扰素受体α链(Interferon-gamma receptor alpha chain,IFN-γRα)、T细胞表达的T盒(T box expressed in T cells,T-bet)、GATA结合蛋白3(GATA binding protein 3,GATA-3)mRNA的表达;蛋白免疫印迹法(Western blotting)检测IFN-γRα、T-bet、GATA-3蛋白的表达;酶联免疫法(Enzyme-linked immunosorbent assay,ELISA)检测细胞培养液上清γ干扰素(Interferon-gamma,IFN-γ)、白细胞介素4(Interleukin-4,IL-4)的表达。对IFN-γRα与IFN-γ、IL-4的表达进行直线相关性分析。结果:流式细胞检测结果显示,miRNA-155模拟体组的IFN-γ阳性CD4+T淋巴细胞计数较对照组显著增加[(63.19±8.61)% vs (47.17±10.28)%,P<0.01] ,阻遏物组较miRNA-155模拟体组降低[(52.87±10.05)% vs (63.19±8.61)%,P=0.024]。三组间IL-4阳性CD4+T淋巴细胞计数无明显差异(F=0.228,P=0.798)。与对照组比较,miRNA-155模拟体组T-bet mRNA表达显著增加(P<0.01),与miRNA-155组比较,阻遏物组T-bet mRNA表达降低(P<0.01);三组间IFN-γRα、GATA-3的mRNA表达无明显差异(分别F=1.055,P=0.362; F=1.601,P=0.220)。与对照组比较,miRNA-155模拟体组IFN-γRα蛋白表达显著减少,T-bet蛋白表达显著增加(均P<0.01);与miRNA-155模拟体组比较,阻遏物组IFN-γRα蛋白表达增加,T-bet蛋白表达减少(均P<0.01);三组间GATA-3蛋白表达无明显差异(F=0.098, P=0.907)。与对照组比较,miRNA-155模拟体组的IFN-γ显著增高(P<0.01),阻遏物较miRNA-155模拟体组降低(P<0.01);三组间IL-4表达无明显差异(F=0.384,P=0.685)。相关分析表明,IFN-γRα蛋白表达与IFN-γ表达呈显著负相关(r=-0.775,P<0.01),与IL-4表达无相关关系(r=0.041,P=0.832)。结论:miRNA-155主要通过影响Th1细胞的分化和功能,参与对CD4+T淋巴细胞的调控,在UAP发病中起重要的作用。miRNA-155的作用可能部分与调控靶基因IFN-γRα的表达有关。目的:初步探讨异常表达miRNA-155与UAP患者在PCI术后心肌损伤关系,为进一步阐明miRNA在PCI术后心肌损伤机制中的作用提供理论基础。方法:选取2010年1月至12月入住我院并行PCI的UAP患者41例,其中PCI术后肌钙蛋白升高超过正常参考值3倍20例,肌钙蛋白正常者21例,同时以18例疑似不典型冠心病症状而冠脉造影正常的住院患者为正常对照组。Real time PCR检测患者循环血CD4+T淋巴细胞miRNA-155在PCI术前和术后12h的表达水平,western blotting检测术前和术后12h IFN-γRα蛋白的表达,ELISA法测定PCI术前和术后12h血清IFN-γ和IL-4的表达。对miRNA-155的表达水平和IFN-γRα蛋白、血清IFN-γ和IL-4进行相关性分析。结果:UAP患者术前miRNA-155、IFN-γ明高于对照组(均P<0.01),IFN-γRα蛋白表达低于对照组(均P<0.01),IL-4表达无差异(均P>0.05)。术前肌钙蛋白阳性组miRNA-155、IFN-γRα蛋白和IFN-γ表达水平与肌钙蛋白阴性组无明显差异(均P>0.05);术后12小时,肌钙蛋白阳性组与肌钙蛋白阴性组的miRNA-155、hsCRP、IFN-γ表达均显著增高(均P<0.05),且肌钙蛋白阳性组较肌钙蛋白阴性组增高更为明显(P<0.05);IFN-γRα蛋白表达显著低于(均P<0.01),肌钙蛋白阳性组较肌钙蛋白阴性组降低更为明显(P<0.01)。两组术前、术后血清IL-4水平无明显差异。术前、术后miRNA-155表达与IFN-γ呈显著正相关(分别r=0.649,P<0.01;r=0.682,P<0.01),与IFN-γRα蛋白表达呈显著负相关(分别r=-0.536,P<0.01;r=-0.592,P<0.01),与IL-4无明显相关关系(分别r=-0.165,P=0.303;r=0.107,P=0.506)。结论:miRNA-155参与了UAP患者中Th1细胞活化及细胞因子IFN-γ分泌的调控过程;miRNA-155表达与PCI术后心肌损伤存在明显的相关关系,miRNA-155可能参与了PCI术后心肌损伤的免疫调控过程。更多还原

【Abstract】 Atherosclerosis is a chronic inflammatory response and autoimmune diseases, and the main pathological basis of coronary heart disease.The previous studies have shown that a large number of T lymphocytes can be found in atherosclerotic plaques, which mainly CD4+T lymphocytes. The imbalance of CD4+T cell subsets closely related with acute coronary syndrome. A large of number activated T lymphocytes in vulnerable plaque resulted in the reduction of plaque stability, which leads to plaque rupture easily. In patients with unstable angina and acute myocardial infarction, platelet thrombus memory has been found in myocardial microvascular and the formation of microembolism in the myocardial microcirculation in, leading to myocardial microinfarction. Thus, The CD4+T lymphocytes may be indirectly involved in the process of myocardial injury in patients with acute coronary syndrome.Myocardial injury after percutaneous coronary intervention remains a common complication, but its mechanism is not fully understood. Some studies have shown that preoperative dose of statins can effectively reduce the levels of inflammatory factors and protect myocardium. Research also shows that, The injury of the vessel wall related to the operation can lead to significant activation of peripheral blood lymphocytes and products of oxidative stress markers was significantly higher.The expression of CD18 in activated CD4+T cells of peripheral blood can promote the recruitment of leukocytes and adhere to the damaged endothelial cells. Therefore, the inflammatory response and the CD4 + T cell-mediated immune response may be directly involved in the process of myocardial injury after percutaneous coronary intervention.MicroRNA is a class of the small molecule RNA about 18 to 25 nucleotides. The recent studies confirm that miRNA are also involved in the regulation of monocytes/macrophages, lymphocytes and endothelial cells, which was closely related with development of atherosclerosis. Although the regulation of miRNA in T lymphocytes has made significant progress, but is still in its infancy.In different disease states and different stages of disease development, the miRNA expression and function varies. The function of miRNA in the regulation of T cell activation in the UAP and myocardial injury after PCI in is still unknown.In the present studies, the miRNA gene chip technology was adopted to screen abnormal expression of the miRNA in CD4+T lymphocytesh in patients with UAP. To observe the effect of abnormal miRNA expression on the CD4+T cell activation, differentiation. The changes of miRNA after PCI and the correlation with myocardial injury were also explored.Objective: To screen differential microRNA (miRNA) expression profiles of CD4+T lymphocyte from the patients with unstable angina pectoris (UAP) and the healthy controls by microarray analysis technique. To elucidate the mechanism responsible for modulation of CD4+T lymphocyte and provide insights into the effects of miRNA on UAP.Methods: Three patients with UAP were enrolled in the study, and three patients with normal coronary artery angiogram were served as a control group. Blood samples were taken from peripheral vein and the CD4+T lymphocytes were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human peripheral blood by magnetic cell sorting system (MACS). The purity of CD4+ T lymphocytes was measured by flow cytometry analysis. The viable count was detected by the rejection experiment of trypanblau. Total RNA was abstracted from CD4+T lymphocyte with Trizol reagent. MiRNA was isolated and enriched of by use of Polyethylene Glycol from 40μg total RNA. The miRNA extracted from CD4+T lymphocytes was hybridized and miRNA expressions profiles of CD4+T lymphocyte were screened with the Affymetrix GeneChip miRNA array. The image signal was scanned by Affymetrix GeneChip Scanner 3000 and analysised by Affymetrix GeneChip Command Console? 1.1 software. Then the image signal was transformed into digital information, which was analysised with SAM software. The differentially expressed miRNA were identified between the two groups. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to confirm the result of selected genes from microarray analysis.Results: The results showed that the expression of miR-155, miR-21, miR-424 and miR-127-3p were over 1.5 folds up-regulated, and the expression of miR-30b and miR-181a were over 0.5 folds down-regulated in UAP group compared to the control group. The qRT-PCR results were in accordance with those obtained using microarray analysis.Conclusion: The differentially expressed miRNA of CD4+T lymphocyte may participate in the the occuring and developing of UAP.KEY WORDS: Unstable angina pectoris;Lymphocyte;microRNA;Gene chip;Real-time polymerase chain reactionObjective: To investigate the effect of abnormal miRNA-155 expression on the differentiations and functions CD4+T lymphocyte in patients with unstable angina pectoris.Methods: Ten patients with UAP were enrolled in the study. Blood samples were taken from peripheral vein. The CD4+T lymphocyte were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human peripheral blood by magnetic cell sorting system (MACS). The CD4+T cells (2×106 cells/ml) were seeded in culture plates of 6 wells. Each well contained 2ml RPMI-1640 medium without 10% fetal bovine serum (FBS). There are four group CD4+T lymphocytes in the experiment: control group (transfected with 50nM N.C using Lipofectamine 2000), miRNA-155 group (transfected with 50 nM miRNA-155 using Lipofectamine 2000), miRNA-155 inhibitor group (transfected with 50 nM miRNA-155 and miRNA-155 inhibitor using Lipofectamine 2000), and FAM-siRNA group (transfected with FAM-siRNA). After stimulated with phytohemagglutinin, the CD4+T lymphocytes and culture supernatant were collected for the following experiments. The frequencies of Th1 and Th2 cells were measured by flow cytometry analysis (FACS). The total RNA and protein were extracted from CD4+T lymphocytes using Trizol and cell lysis buffer for western blotting, respectively. The level of IFN-γRα、T-bet、GATA-3 mRNA expression were measured by qRT-PCR. The level of IFN-γRα、T-bet、GATA-3 protein expression were examined using western blotting. The productions of IFN-γand IL-4 in culture superatants of CD4+T lymphocytes were detected by enzyme-linked immunosorbent assay (ELISA). Pearson correlation analysis was conducted to examine the association between IFN-γRαand IFN-γ, IL-4.Results: The FACS showed that miRNA-155 could promote CD4+T lymphocytes to Th1 cells and the Th1 frequencies were also significantly increased in miRNA-155 group compared with the control group and miRNA-155 inhibitor group[(63.19±8.61)% vs (47.17±10.28)%,P<0.01;(63.19±8.61)% vs (52.87±10.05)%,P=0.024,respectively]. There was no significant difference between the three groups in the frequencies of Th2 cells (F=0.228,P=0.798). In comparison with the control group, there was significant increase in the level of T-bet mRNA expression in miRNA-155 group (P<0.01). The level of T-bet mRNA expression in miRNA-155 inhibitor group was significantly lower than that in miRNA-155 group (P<0.01). No significant differences were found between the three groups in the level of IFN-γRαand GATA-3 mRNA expression (F=1.055, P=0.362; F=1.601, P=0.220, respectively). The level of IFN-γRαprotein expression in miRNA-155 group was significantly higher than that in miRNA-155 group and miRNA-155 inhibitor group (all P<0.01). There was no significant difference between the three groups in the level of GATA-3 protein expression(F=0.098, P=0.907).The culture supernatant concentration of IFN-γin miRNA-155 group was significantly increased than that in the control group and miRNA-155 inhibitor group (all P<0.01). No significant difference was observed between the three groups in the culture supernatant concentration of IL-4(F=0.384,P=0.685). There was significant negatibe correlation between the protein expression of IFN-γRαand the level of IFN-γ. No significant correlation were found between IFN-γRαand IL-4.Conclusion: It is evident that miRNA-155 can promote Th2 cells differentiations, and thus improving the function, which partly attributed to inhibit the expression of IFN-γRαprotein and may play an important role in the pathogenesis of unstable angina.Objective: To study the relationship of abnormal expressed miRNA-155 of CD4+T lymphocyte and myocardial injury after percutaneous coronary intervention in patients with unstable angina. To provide insights into the effects of miRNA-155 on myocardial injury in patients undergoing PCI.Methods: A total of 41 individuals with UA, who presented to the cardiology department of the First Affiliated Hospital of Guangxi Medical University were enrolled from January 2010 to December 2010. The patients were divided into two groups based on their troponin I (TnI) levels; elevated TnI group (TnI>0.15ng/ml, 20 cases) and normal TnI group (TnI≤0.15ng/ml, 21cases). Eighteen normal control subjects who were confirmed by coronary angiography were enrolled in the study. The level of miRNA-155 expression was measured by qRT-PCR before PCI and 12h after PCI. The level of IFN-γRαprotein expression were examined using western blotting and the level of serum IFN-γand IL-4 were detected by ELISA before PCI and 12h after PCI. Correlation analysis was made between miRNA-155 and the level of IFN-γRαprotein,serum IFN-γand IL-4.Results: Results: Compared with the control group, the expression of miRNA-155 and IFN-γwere significantly increased (all P<0.01), and the level of IFN-γRαwas markly decreased in the elevated TnI group and the normal TnI group before PCI (all P<0.01). There was no significant different in the level of IL-4 between the three groups before PCI (P>0.05). There was no significant different in the expression of miRNA-155、IFN-γRαand IFN-γbetween the elevated TnI group and the normal TnI group before PCI (all P>0.01). The expressions of miRNA-155, hsCRP, IFN-γwere significant increased, and the level of IFN-γRαprotein was decreased in the elevated TnI group and the normal TnI group at 12h after PCI (all P<0.05). The changes were more obvious in the elevated TnI group than the normal TnI group (all P<0.05). There was no significant different in the level of IL-4 in the elevated TnI group and the normal TnI group before and after PCI in. The expressions of miRNA-155 were significantly positive with IFN-γ(r=0.649 , P<0.01 ; r=0.682 , P<0.01, respectively), and significantly negative with IFN-γRαbefore and after PCI(r=-0.536 , P<0.01 ; r=-0.592 , P<0.01, respectively). There was no correlation between miRNA-155 and IL-4 before and after PCI (r=-0.165, P=0.303;r=0.107,P=0.506, respectively).Conclusion: The evidence showed that abnormal expressed miRNA-155 of CD4+T lymphocyte participated in the regulation process of Th2 cells differentiations, and function. There was correlation between abnormal expressed miRNA-155 and myocardial injury after PCI. MiRNA-155 may involve in the immunoregulation process of myocardial injury after PCI.更多还原