【作者】 陶丹萍；

【导师】 李志樑；

【作者基本信息】 南方医科大学 ， 内科学， 2011， 硕士

【摘要】 冠心病(coronary heart disease CHD)是20世纪以来威胁人类生命健康的最主要疾病之一,而且其发病率越来越高,其发病原因及防治方法一直是社会关注的焦点。冠心病的诊治方法日渐完善,但其发病机制却未十分明了。动脉粥样硬化(atherosclerosis AS)是心脑血管疾病发病的主要病理基础。AS的形成机制学说主要有脂质沉淀、损伤修复、血栓形成等。众所周知,心脏病家族史、性别、高龄、吸烟、高血压病、糖尿病、高脂血症等是AS的高危因素,近年研究表明感染因素如肺炎衣原体、幽门螺旋杆菌、巨细胞病毒等也与CHD有重要关系。急性冠脉综合征是冠心病的一个重要进展阶段,而易损斑块是急性冠脉综合征的主要病理基础。国内外的研究发现,炎症反应在易损斑块的进展中发挥着重要的作用,是斑块不稳定及容易破裂的主要原因,因而CHD可能是一种冠状动脉慢性炎症及全身免疫性疾病。树突状细胞(Dendritic cell, DCs)在免疫过程中主要起抗原呈递作用,是体内功能最强的专职抗原呈递细胞。极少量的DCs即可强烈激活T淋巴细胞启动特异性细胞免疫反应。国内外研究发现,正常动脉内膜存在一种血管相关淋巴组织(VALT),散在分布着一些由免疫活性细胞和抗原呈递细胞组成的细胞群,对血管组织中可能有害的内源性或外源性抗原进行监视和筛查。研究发现,DCs在AS病变中聚集明显增加,DCs与T淋巴细胞共同出现于AS的病变薄弱位置。当DC遇到内源性或外源性抗原时,可将抗原呈递给T细胞,使其活化增殖,分泌细胞因子,然后启动一系列免疫反应,导致AS的发生发展。最近的研究显示氧化修饰的低密度脂蛋白可通过激活DCs介导的获得性免疫反应,促进AS病变的进展。研究发现,氧化型低密度脂蛋白(oxidized low density lipoprotein, ox-LDL)是一种内源性免疫反应激活剂,可引起血管内皮功能失调、参与泡沫细胞形成、促进血管平滑肌细胞增殖及诱导凋亡等。已有的研究表明,ox-LDL在动脉粥样硬化局部沉积后,促进DC与血管内皮粘附,并且作为自身抗原刺激局部产生抗氧化型低密度脂蛋白抗体,促进DC分化成熟,并激活T淋巴细胞,参与动脉粥样硬化局部免疫炎症病变。适度ox-LDL可促进单核细胞来源的树突状细胞成熟,增强其激活T细胞的功能,增加DC分泌细胞因子IL-2、IL-12、IL-18、INF-γ(?)(?)TNFα等,并且这些作用随ox-LDL浓度和氧化程度的增高而增强,但是过高的浓度却会导致DCs凋亡越来越多研究发现,多种炎症标志物参与了CHD的发生、发展及预后,如IL-6、IL-10、IL-18、IL-12及超敏C反应蛋白(high sensitive C-reactive protein, hs-CRP)等。IL-27 (Interleukin-27, IL-27)是新近发现的属于IL-6/IL-12家族细胞因子,由p28和EBI3组成,具有促进及抑制免疫反应的双重作用。由于IL-27在免疫反应和免疫耐受的调节中扮演了不可或缺的角色,因此在炎症性、自身免疫性、感染性、肿瘤性疾病中均发现IL-27的表达及功能变化。但是在冠心病的研究中却十分稀少,本研究通过检测不同类型冠心病患者的血浆IL-27水平,了解CHD患者血浆IL-27的变化,并探讨它们之间的关系。IL-27主要由活化的DC细胞产生,单核细胞、巨噬细胞、NK细胞等也可以分泌一定量的IL-27,当DCs表面的Toll样受体(TLRs)受到病原体相关分子模式(PAMP)刺激后会分泌IL-27。人外周血单核细胞来源的DC表达IL-27受体(EBI3和p28组成的异二聚体),提示IL-27能够对单核细胞来源的DC产生影响,它可以反馈作用于DCs,促进DCs表面共刺激分子的表达以及提高其激活辅助性T细胞(Th)的能力。国外研究学者发现WSX-1敲除的小鼠中DC在体外受到LPS刺激后表面分子CD80/CD86表达上调,诱导Thl细胞增殖和产生IFN-γ的能力也大大提高。DCs是联系天然防御功能和获得性免疫的关键,与CHD发生发展密切相关,那么在CHD发生发展的机制中,IL-27是否通过作用于DCs发挥免疫调节功能?IL-27处理后DCs的功能状态如何,是否会影响其功能蛋白及免疫调节分子的分泌?本文旨在研究IL-27对体外扩增DC免疫活性的影响,为IL-27结合DC用于CHD免疫机制研究提供依据。本课题分为三个部分,第一部分：通过ELISA法及免疫比浊法分别检测不同类型冠状动脉粥样硬化性心脏病(简称冠心病,CHD)患者血浆白介素27(IL-27)及超敏C反应蛋白(hs-CRP)的水平,并进行比较,以探讨IL-27是否与冠心病的进展以及粥样斑块的稳定密切相关。第二部分：将人PBMC来源的DC作为实验模型,以OX-LDL作为抗原诱导DC成熟,用RT—PCR的方法测定DC产生IL-27亚基的情况,探讨人体内IL-27的由来。第三部分：将人PBMC来源的DC作为实验模型,以OX-LDL作为抗原诱导DC成熟,用流式细胞技术及ELISA方法,观察DC的成熟程度和分泌细胞因子的能力,并进一步应用IL-27进行干预研究。本课题通过研究IL-27对体外扩增DC免疫活性的影响,为IL-27结合DC用于CHD免疫机制研究提供依据,为AS发生发展的免疫学介导学说提供实验依据,具有深远的意义。结果分述如下：1不同CHD患者血浆中IL-27及hs-CRP水平具有差异性1.1入选各组病例一般临床资料比较无显著性差异临床入选病例共88例,其中对照组28例,SAP组10例,UAP组25例,AMI组25例,以上各组一般临床资料的比较无显著性差异,具有可比性。1.2不同CHD患者血浆中IL-27水平具有差异性CHD组血浆IL-27水平明显高于对照组；SAP组及UAP组血浆IL-27水平无差异；UAP组及SAP组血浆IL-27水平高于AMI组。1.3不同CHD患者血浆中hs-CRP具有差异性CHD组患者血浆hs-CRP水平高于对照组；UAP组血浆hs-CRP水平高于SAP组；AMI组均高于UAP及SAP组。1.4 hs-CRP、IL-27在CHD中表达的关系将88例患者血清中hs-CRP与IL-27进行相关分析,结果显示IL-27与hs-CRP呈正相关,r=0.308,P<0.01。2 ox-LDL诱导人DC细胞成熟,用RT-PCR的方法测定DC的IL-27亚基mRNA表达将加ox-LDL分为四组(n=3)：(1)对照组：DC+PBS；(2)DC+ox-LDL(100mg/L,作用8小时)组；(3)DC+0x-LDL(100mg/L,作用16小时)组；(4)DC+ox-LDL(100mg/L,作用24小时)组。2.1 DC的镜下鉴定：倒置相差显微镜及扫描电镜下,从健康人外周血中分离的PBMC经ox-LDL诱导后成为细胞形态不规则、表面毛刺状突起、细胞质丰富的成熟DC。2.2用RT-PCR的方法检测DC的IL-27亚基(P28及EBI3)mRNA表达ox-LDL上调DC的IL-27亚基(P28及EBI3)mRNA的表达,ox-LDL刺激组P28及EBI3mRNA表达均比对照组高,并有时间依赖性的特点。3 IL-27对ox-LDL介导树突状细胞免疫成熟作用的研究将加IL-27分为三组(n=5),均作用24小时：(1)对照组：DC+PBS；(2)DC+ox-LDL(100mg/L组；(3)DC+ox-LDL(100mg/L)+IL-27(100ng/mL)组。3.1 DC的镜下鉴定：倒置相差显微镜及扫描电镜下观察,从外周血中分离的PBMC经ox-LDL及IL-27诱导后成为细胞形态不规则、表面毛刺状突起、细胞质丰富的成熟DC。3.2用流式细胞仪检测对DC细胞表型的影响3.2.1 ox-LDL上调DC细胞表型表达流式细胞仪检测人外周血PBMC来源的DC表型HLA-DR.CD83及CD86。ox-LDL组HLA-DR.CD83及CD86表型表达较对照组高,说明ox-LDL可刺激外周血PBMC来源的DC成熟,具有一定的抗原呈递功能。3.2.2 IL-27上调ox-LDL诱导成熟的DC表型的表达加入IL-27后DC表面的HLA-DR、CD83及CD86表达高于ox-LDL组,说明IL-27可进一步促进ox-LDL刺激外周血PBMC来源的DC成熟。3.3对DC培养上清液细胞因子IL-6、IL-10及IL-12p70的影响3.3.1 ox-LDL上调DC培养上清液细胞因子IL-6及IL-10的表达,下调IL-12p70的表达。ox-LDL组DC培养上清液细胞因子IL-6及IL-10的表达均比对照组高,IL-12p70表达比对照组低。说明ox-LDL刺激DC免疫成熟后,促进Thl型细胞因子IL-6分泌,具有Th1型免疫反应的能力。3.3.2 IL—27上调ox-LDL诱导成熟的DC表达IL-6,下调IL-10的表达。用ox-LDL诱导DC成熟后,加入IL-27刺激,DC培养上清液细胞因子IL-6表达比ox-LDL高,IL-10的表达比ox-LDL低。说明IL-27具有一定促进Thl型免疫反应及抑制Th2型免疫反应的能力。通过上述三个部分的实验,我们可以得出以下结论：1、不同类型CHD患者血浆IL-27及hs-CRP均较对照组升高,说明与病情相关,可作为CHD病情评估的参考指标；2、ox-LDL上调DC的IL-27亚基(P28及EBI3)mRNA的表达,说明ox-LDL能够使活化的人PBMC来源的DC分泌IL-27。3、ox-LDL可促进DC免疫分化成熟。4、ox-LDL可促进DC免疫成熟,并促进DC分泌IL-6及IL-10。5、IL—27上调ox-LDL所诱导的DC的免疫成熟,并进一步促进DC分泌IL-6及抑制DC分泌IL-10。更多还原

【Abstract】 Coronary heart disease (CHD) has become one of the greatest threat -ens to human beings since the 20th century. And its incidence increases gradurily when its causes and control methods has been the focus of social concern. The diagnosis and treatment of CHD is more and more, but its pathogenesis has not been very clear. Atherosclerosis (AS) is the main pathology of the cardiovascular disease. Lipid deposition, damage repair and thrombosis are the formation mechanism of AS. As we all know, heart disease, family history, gender, age, smoking, hypertension, diabetes and hyperlipidemia are the risk factors of AS. But we found that, the infection factors such as Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus aslo have great relationships with CHD. More and more studies show that acute coronary syndrome(ACS) is an important stage of progress of CHD, and the vulnerable plaque is the main pathology of ACS. Domestic and international study found that inflammation plays an important role in unstable plaque which is the main reason of causing plaque rupture. CHD is a chronic inflammatory and systemic autoimmune disease.The function of Dendritic cells (DCs) in the immune process is antigen presentation,which is the most powerful professional antigen-presenting cell. Very small amount of DCs can activate T lymphocytes to start strong immune response of specific cells. The domestic and international studys found that there is a vessel associated lymphoid tissue (VALT) in the intima where scattered with some immune cells and antigen-presenting cells, monitoring and screening the endogenous or exogenous antigens which may be harmful to the vascular tissue. We found that the numerber of DCs increased significantly in the AS lesions, DCs and T lymphocytes appear together in the weak position of AS. When the endogenous or exogenous antigens appear to DCs, the antigen can be presented to T cells which can activate and proliferate by DCs,and then secreting cytokines and starting a series of immune responses which leading to the development of AS. Recent studies have shown that oxidized low density lipoprotein(ox-LDL) can promote the progress of AS lesions through the acquired immune response activated by DCs.We found that ox-LDL is an endogenous immune response activator which can cause dysfunction of endothelial, involve in the formation of foam cells, promote proliferation of vascular smooth muscle cell and induce apoptosis. Previous studies showed that, ox-LDL which deposes localy in the AS, can promote adhesion between DCs and vascular endotheliar, stimulate the production of anti-oxidized low density lipoprotein antibody as a self-antigen, and promote differentiation and maturation of DC, activate T lymphocytes, and the leading the immune inflammation of local atherosclerotic lesions. Moderate ox-LDL can promote maturation of monocyte-derived DCs and enhance its function of activating T cell, increase IL-2, IL-12, IL-18, INF-y and TNFa secreted by DCs, and these effects are enhanced by the concentration and degree of oxidation of ox-LDL, but the too high concentration of ox-LDL will cause apoptosis of DCs.More and more researchs show that a variety of markers of inflammation involve in the occurrence, development and prognosis of CHD, such as IL-6, IL-10, IL-18, IL-12 and high sensitivity C reactive protein(hs-CRP). IL-27 is a newly discovered cytokines which composied by p28 and EBI3, belonging to IL-6/IL-12 family, which plays dual role in the immune response. The expression and functional changes of IL-27 were found in inflammatory, autoimmune, infectious, and neoplastic diseases because of its vital role of the regulation in immune response and tolerance. However, the study of IL-27 in CHD is very rare. We will explore the relationship between IL-27 and CHD by detecting the plasma level of IL-27 beweet different CHD patients.IL-27 is mainly produced by the activated DCs, monocytes, macrophages, NK cells, also can secrete a certain amount of IL-27. The secretion of IL-27 will happen when the the toll like receptors (TLRs) of DCs stimulating by the pathogen-associated molecular patterns (PAMP). DCs derived from human peripheral blood monocyte(PBMC) express receptor(heterodimer composed of EBI3 and p28) of IL-27, suggesting that IL-27 has an impact on DCs, which has a feedback on the DCs, promoting DCs to express costimulatory molecules and enhancing the activation of T helper cell (Th). Foreign researchers found that the DCs of WSX-1 knockout mice will increase the expression of surface molecules CD80/CD86 after stimulated by LPS in vitro, and then inducing proliferation of Thl cell and improving the production of IFN-γgreatly.DCs contacts the natural and acquired immune defense function, which is closely related to the development of CHD. Can IL-27 affect the function of immune by affecting DCs? How the functional status of DCs will be after the affection by IL-27? Will the function of proteins and secretion of immune regulatory molecules be affacted? This article aims to study the influence of IL-27 on immune activity of DC in vitro,to provide the basis mechanism for CHD.This article is divided into three parts, Part 1:the levels of IL-27 and high sensitivity C-reactive protein (hs-CRP) were detected by ELISA and immune turbidimetry of different types of CHD,which will be compared,with the purpose of investigating whether IL-27 is closely related to the stability of atherosclerotic plaque, and progress of coronary heart disease. Part 2: ox-LDL is used as an antigen to induce DCs maturation, the determination of subunits produced by DC by RT-PCR method, to explore the origin of IL-27. Part 3:ox-LDL is used as an antigen to induce DCs maturation, we observed the extent of DC maturation and secretion of cytokine by flow cytometry and ELISA, and further studies of application by IL-27 intervention. The article provide evidence of the immune mechanism for CHD by the combination of IL-27 and DCs by the influence on immune activity of DC,which provides experimental evidence of the development of AS-mediated immune. The results are summarized as follows:1 the levels of IL-27 and hs-CRP were significantly different among different CHD patients1.1 Comparison of the general clinical data among different groups showed no significant difference.88 cases were divided into as follow: control group (28 cases), SAP group (10 cases), UAP group (25 cases), AMI group (25 cases). Comparison of the general clinical data showed no significant difference among different group.1.2 Peripheral blood levels of IL-27 were significantly different among different CHD patients.Levels of IL-27 of CHD were more than the control group; Levels of IL-27 were the same between SAP group and UAP group; while the levels of IL-27 in SAP group and UAP group were higher than AMI group.1.3 Peripheral blood levels of hs-CRP were significantly different among different CHD patients.Levels of hs-CRP of CHD were more than the control group; Levels of hs-CRP of UAP group were more than the UAP group; while the levels of hs-CRP of AMI group were higher than SAP and UAP group.1.4 The relationship between IL-27 and hs-CRP of CHD.The correlation analysis showed that IL-27 and hs-CRP were positively correlated of 88 cases. 2 ox-LDL is used as an antigen to induce DCs maturation, the determination of subunits produced by DC is evaluate by RT-PCR methodThe test include 4 groups(n=3):(1) Control group:DC+PBS; (2) DC+ox-LDL(100mg/L,8h) group; (3) DC+ox-LDL(100mg/L,16h) group; (4) DC+ox-LDL(100mg/L,16h) group.2.1 Identification of DC by microscopeCell shapes under inverted phase contrast microscope and electron microscope are reported as fllowing:the cells shapes changed from round to irregular after induced by ox-LDL, some spinule-like structures newly erupted and the cytoplasm were getting thicker and thicher.2.2 The determination of subunits produced by DC is evaluate by RT-PCR methodOx-LDL increased the mRNA expression of IL-27 subunits(P28 and EBI3), the mRNA expressions of P28 and EBI3 of ox-LDL are higher than the control group, with the time-dependent characteristics.3 The roles of ox-LDL on DC maturation derived from PBMC immune and intervention by IL-27.The test includes 3 groups(n=3), atction for 24h:(1) Control group: DC+PBS;(2)DC+ox-LDL(100mg/L)group;(3)DC+ox-LDL(100mg/L)+IL-27(100ng/ ml)group.3.1 Identification of DC by microscope.Cell shapes under inverted phase contrast microscope and electron microscope are reported as fllowing:the cells shapes changed from round to irregular after induced by ox-LDL, some spinule-like structures newly erupted and the cytoplasm were getting thicker and thicher.3.2 Impact on the cell phenotypes, identified by flow cytometry.3.2.1 The upregulation of phenotypes expression on DC by ox-LDL. Phenotypes of DC derived from PBMC in peripheral blood were detected by flow cytometry. Phenotypes of DC including CD 83, CD86 and HLA-DR stimulated by ox-LDL are higher than the control group, indicating that ox-LDL can induce DC maturation which has a certain antigen-presenting function.3.2.2 IL-27 increase the expression of DC phenotypes induced by ox-LDL.The expression of DC phenotypes of CD83, CD86 and HLA-DR which post-treated by IL-27 were higher than that in ox-LDL group. This indicate IL-27 may promote the maturation by ox-LDL.3.3 Impact on levels of IL-6, IL-10 and IL-12p70 of DC stimulated by ox-LDL and IL-27.3.3.1 ox-LDL increase the levels of IL-6 and IL-10, decrease the levels of IL-12p70.After co-cultured DC stimulated by ox-LDL, the levels of IL-6 and IL-10 are higer than that in PBS group while the levels of IL-12p70 are lower. This indicate that ox-LDL may stimulate DC maturation, promote Thl secreteing IL-6, and have capability to promote Thl-type immune response.3.3.2 IL-27 increased the levels of IL-6 of DC stimulate by ox-LDL and decrease levels of IL-10.The levels of IL-6 of DC stimulated by ox-LDL which post-treated by IL-27 are higher than those of ox-LDL group while the levels of IL-10 are lower. This indicate that IL-27 may have the capability to promote Thl-type immune response and inhibit Th2-type immune response.The results are reported as follow:①The levels of IL-27 of different CHD patients were significantly higher than control group.This indicate that levels of IL-27 is associated with the disease which can be reference for assessment of CHD. ②Ox-LDL increased the mRNA expression of IL-27 subunits(P28 and EBI3). This indicate that ox-LDL can promote the secrection of IL-27 of DC derived from PBMC.③Ox-LDL can promote the maturation and differentiation of DC.④O-LDL can promote the maturation and differentiation of DC, and has the capability to promote the secretion of IL-6 by DC.⑤IL-27 may promote the maturation by ox-LDL and have the capability to promote the secretion of IL-6 by DC and inhibit the secretion of IL-10 by DC.更多还原