【作者】 陈朝俊；

【导师】 李志樑；

【作者基本信息】 南方医科大学 ， 心血管内科， 2009， 博士

【摘要】 冠心病（coronary heart disease,CHD）作为进入二十世纪以来,威胁人类生命健康的主要疾病,其有效防治一直成为整个社会关注的重点和难点。但是CHD的发病机制至今仍不明了,成为阻滞其治疗进展的主要难题。动脉粥样硬化（atherosclerosis,AS）是心脑血管疾病的发病的主要病理基础。以往关于AS形成的机制主要围绕三种学说,脂质沉淀学说、损伤修复学说和血栓形成学说。因而,高龄、血脂升高、吸烟、血压升高、血糖升高、高半胱氨酸血症以及高尿酸血症等导致血管内皮损伤和脂质沉积的内在因素,被界定为CHD发病的高危因素。远离这些危险因素,或者控制这些因素的恶化,在一定程度上取得了预防AS发生发展的成果。然而,随着CHD发病的逐渐年轻,进展逐渐加快,使得人们对危险因素的预防治疗提出了疑问。近年来,随着急性冠脉综合症（acutecoronary syndrome,ACS）这个CHD的急性进展阶段的界定,使得人们重新认识了CHD发展阶段的病理和病理生理特点。研究发现ACS的病理基础是易损斑块,并且发现易损斑块伴随着大量炎症细胞和免疫细胞的介入,是斑块不稳定和容易破裂的主要原因;ACS患者存在全身炎症因子和炎症反应活化的情况。进而,发现AS的所有阶段都有免疫细胞的介入,提出CHD是一种慢性炎症和自身免疫性疾病这一新观点,认为内在或外在危险因素导致的炎症和免疫反应是AS发病的基础。树突状细胞（Dendritic cells,DCs）是免疫过程中起负责抗原呈递的细胞,是机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,其激活T淋巴细胞的能力是巨噬细胞或B细胞的100～1000倍,在免疫应答的诱导和调节中起重要作用。最近研究证实在动脉壁中有引起免疫反应发生的微环境,正常动脉内膜存在一种血管相关淋巴组织（Vascular-associated lymphoid tissue,VALT）,类似于呼吸道和胃肠道中的粘膜相关淋巴组织,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。VALT中发现有血管树突状细胞（Vascular dendritic cells,VDCs）的存在,正常的动脉壁中VDCs较少,属未成熟形式,主要分布在血管的内膜和外膜。VDCs在AS病变中聚集明显增加,在炎症浸润区域往往与T淋巴细胞和巨噬细胞聚集在一起。DCs与T淋巴细胞共同出现在AS的病变薄弱部位,使得人们推测DCs在AS的发生发展中可能同样起着启动炎症和免疫反应的作用。最近的研究显示氧化修饰的低密度脂蛋白和尼古丁可通过激活DCs介导的获得性免疫反应,促进AS病变的进展。说明DCs可能参与了AS的病理发生,在触发和放大AS炎症免疫反应中起重要的调节作用。黄芪多糖（Astragalus polysaccharids,APS）是黄芪中重要的天然有效成分之一,具有双向调节血糖、促进免疫、提高巨噬细胞活性、促进淋巴细胞转化,激活T细胞B细胞并能促进抗体的形成,从而抗击各种病毒性和细菌性疾病,此外,研究发现APS在多种疾病中发挥重要作用,如APS具有抗肿瘤作用,但体外实验表明APS不能有效抑制肿瘤细胞生长,推测其抗肿瘤作用可能与APS的免疫诱导作用有关。APS可显著降低非肥胖小鼠1型糖尿病的发病率,保护胰腺小岛的超微结构,恢复Th1细胞群和Th2细胞群间平衡。APS能够降低SD大鼠肾皮质NF-κB mRNA水平的表达,提高IκB mRNA的表达,可有效的防治糖尿病肾病的进展。APS能够有效抑制金属诱导的氧化应激,抑制肾脏的缺血再灌注损伤,调控免疫反应,抑制肾小球系膜细胞增殖和基质的过度合成以及下调肾小球系膜细胞β1整合素的表达。在心血管疾病相关研究中,APS能有效降低健康人总胆固醇、载脂蛋白H、低密度脂蛋白的水平,对50岁以上中老年健康人群的三酰甘油水平也同样有效。在对糖尿病心肌病仓鼠模型的研究中,APS通过抑制仓鼠局部心肌糜蛋白酶-AngⅡ系统发挥抗仓鼠糖尿病心肌病的作用。此外,有研究表明APS是通过抑制血小板钙调蛋白而抑制磷酸二酯酶的活性,从而增加血小板内cAMP含量,抑制血小板聚集和血栓形成。此外,APS是黄芪注射液的主要成分之一,在对冠心病心绞痛的治疗中,黄芪注射液能明显减少心绞痛的发作次数,延长发作间歇时间,临床疗效改善显著。黄芪注射液能够干预CHD发生发展可能与APS的免疫调节功能密不可分。由于DCs作为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治疗中,APS是否通过作用于DCs发挥免疫调节功能?APS处理后具有抗原呈递功能的DCs功能状态如何,是否会影响其功能蛋白和免疫调节分子的分泌,是否会影响这些蛋白的基因表达。这些问题中,研究APS处理后DCs的基因变化是问题关键。本课题分为三个部分,将分离的临床人外周血单核细胞（peripheral bloodmononuclear cell,PBMC）和血清为研究对象,以研究APS处理后外周血PBMC源性DCs基因表达和功能变化为切入点,通过免疫荧光、流式细胞术、基因芯片、RT-PCR和ELISA等方法,重点观察APS处理外周血PBMC源性DCs的成熟状态、免疫功能和其他基因表达的差异与AS发生发展的关系,深入探讨APS处理后抗原呈递细胞功能变化在AS治疗过程的地位和作用。旨在为AS发生发展的免疫介导学说提供实验依据和免疫治疗途径,为心脑血管疾病的防治提供新的理论平台和技术手段,具有长远的经济价值和重要的社会意义。结果分述如下:1.APS处理后人外周血来源的DCs处于成熟状态,并具有诱导T细胞增殖的能力1.1人外周血来源的DCs培养与鉴定培养第2天对照组细胞粘附于聚苯乙烯培养瓶底部,实验组的细胞部分在培养基中悬浮生长,细胞较小,形态无明显差别。培养第6天对照组的细胞呈圆形或椭圆形,大部分粘附于培养瓶瓶底;LPS组、100mg/L APS组的细胞生长状态最好,呈悬浮生长,可见部分细胞聚集成簇,细胞表面可见数量不等、形态不一的树突样突起。200mg/L APS组多数细胞胞质内可见大量空泡样结构,可见细胞崩解,有大量不定型的细胞碎片。流式细胞仪检测APS100mg/L组人外周血PBMC来源的DCs表型为CD1α⁺,CD80⁺,CD83^low,CD86⁺,HLA-DR⁺。1.2 DCs培养上清液细胞因子IL-12检测结果因200mg/L组细胞大量凋亡崩解,故选择对照组、LPS组、50mg/L APS组、100mg/L APS组细胞上清液中IL-12进行检测。培养6天后,4组细胞上清液中IL-12含量存在显著性差异（P=0.000）;其中,LPS组、100mg/L APS组细胞上清液中IL-12含量分别与对照组、50mg/L APS组相比存在显著性差异（P=0.000）,明显高于后两组。1.3各组外周血PBMC来源的DCs表型变化情况选择对照组、50mg/L APS组、100mg/L APS组、LPS组进行流式细胞学检测。100mg/L APS组、LPS组DCs表面CD1α、CD86等表型表达上调,与对照组、50mg/L APS组相比,存在显著性差异（P＜0.05）;LPS组HLA-DR表达与50mg/L APS组相比,存在显著性差异（P＜0.05）,50mg/L APS组HLA-DR表达较低。而各组DCs表面CD83表型表达各组之间差异无显著性意义。1.4各组DCs的同种混合淋巴细胞反应³H-TDR掺入率在4组间存在显著性差异（P＜0.01）。DCs在与T淋巴细胞混合比例在1∶50、1∶20时,100mg/L APS组、LPS组的³H-TDR掺入率与其他两组相比,存在显著性差异（P＜0.01）,较其余两组明显升高;而对照组、50mg/LAPS组之间的³H-TDR掺入率相比,无显著性学差异（P＞0.05）。2.基因芯片检测外周血PBMC来源的DCs基因表达不同芯片检测的结果显示,APS处理后人外周血DCs功能蛋白表达基因中有7个基因呈明显上升,分别是CD36、IGSF6、IL27、INHA、LIPA、MAP4K3和SOD2,分别属于抗原识别受体、细胞因子、细胞酶类和细胞内信号传导系统。有26个基因呈明显下降表达,分别是ADAM19、ADAR、BASP1、BTG1、CCR7、CD40LG、CST7、CXCL9、CXCR4、DCTN2、EBI3、ISG15、ICAM1、IFI16、IL15、ISG20、CD207、LTA、MARCKS、MARCKSL1、NFKB1、NFKB2、PNRC1、RAC1、RELB和TNFRSF11B等,分别属于细胞酶类、抗原识别受体、细胞趋化因子受体、细胞因子和细胞内信号传导系统。APS处理后人外周血PBMC来源的DCs分别在细胞粘附及趋化DCs迁徙的受体及相关蛋白如ADAM19、CCR7、CD40LG、CST7、CXCL9和CXCR4等的表达减低;而与诱导细胞增殖及活化相关的信号传导通路及功能蛋白的基因表达如ADAR、BASP1、BTG1、DCTN2、ISG15、ISG20、MARCKS、MARCKSL1、NFKB1、NFKB2、PNRC1、RAC1、RELB和TNFRSF11B也同样下降;而与抗原呈递及诱导免疫反应相关蛋白的基因表达如EBI3、IFI16、IL15、CD207和LTA等表达下降。而作为升高的表达基因,有抗原结合蛋白基因如CD36;有消化及降解抗原的相关细胞及溶酶体酶类蛋白如LIPA和SOD2;同样有抑制炎症及免疫反应的蛋白基因表达增多,如IL27和INHA等。3.荧光定量PCR检测基因芯片结果,显示DCs基因芯片检测有较好的可信度应用荧光定量PCR检测芯片检测表达上调的基因CD36和IL-27,以及表达下调的基因FIF16表达情况显示,APS处理后DCs组CD36和IL-27基因表达较对照组增加幅度与基因芯片检测相似;而FIF16基因表达较对照组降低加幅度与基因芯片检测相似。通过上述三个部分的实验,我们能够得出以下结论:①APS处理人外周血PBMC源的DCs细胞形态及表型表达处于成熟状态;②APS处理人外周血PBMC源的DCs,分泌IL-12水平较对照组明显升高,且具有诱导同种T淋巴细胞增殖的能力;③APS处理后人外周血DCs功能蛋白表达基因中有7个基因呈明显上升,分别是CD36、IGSF6、IL27、INHA、LIPA、MAP4K3和SOD2,分别属于抗原识别受体、细胞因子、细胞酶类和细胞内信号传导系统。有26个基因呈明显下降表达,分别是ADAM19、ADAR、BASP1、BTG1、CCR7、CD40LG、CST7、CXCL9、CXCR4、DCTN2、EBI3、ISG15、ICAM1、IFI16、IL15、ISG20、CD207、LTA、MARCKS、MARCKSL1、NFKB1、NFKB2、PNRC1、RAC1、RELB和TNFRSF11B等,分别属于细胞酶类、抗原识别受体、细胞趋化因子受体、细胞因子和细胞内信号传导系统。④荧光定量PCR检测芯片检测表达上调的基因CD36和IL-27,以及表达下调的基因IFI16表达情况,验证了芯片检测的结果。更多还原

【Abstract】 Coronary heart disease（CHD）has been the main threaten to human health since the arrival of 21th century.The effective prevention and treatment of CHD has being the emphasis and challenge of the whole society of human beings.However,the pathogenesis of CHD has not been clearly identified so it is a great hindrance to the clinical treatment of CHD.Atherosclerosis（AS）is the pathologic basis of cardiac cerebro vascular diseases.In the past,there were mainly 3 theories,including lipidose deposition,injury repair and thrombogenesis,to explain the forming of AS.On basis of these theories,factors such as aging,hyperlipemia,hypertension,hyperglycaemia, hypercysteinemia and high blood uric acid are termed as high risks of CHD because they may induce blood vessel endothelium injury and lipidose deposition.Some progresses have been made in prevention of the genesis and advancement of AS by getting rid of or controlling of these high risks.However,people are beginning to question the effects of the preventions and treatments on these high risks for that the morbility age of CHD are younger than ever before,and the progress of AS speeds up. With the terming of ACS on acute progress stages of CHD,People re-recognized the pathologic and pathophysiologic characteristics of CHD in their developmental stages. Studies on AS found that unstable plaques are the pathological basis of ACS.The interventions of the inflammatory cells and immunocells to unstable plaques are key factors that leading to the instability and easily rupture of AS plaques.It is considered that systemic activation of the inflammatory factors and inflammatory reactions exist in ACS cases.What’s more,people found that immunocells involve in all the AS developmental stages.They had proposed a new point of view that CHD is a process of chronic inflammation and a disease of autoimmunity.It is considered that inflammatory reactions and immune reactions induced by endogenous and exogenous risks are the pathological basis of AS.Dendritic cells（DCs）are the most powerful specific antigen presenting cells that play a role of presenting antigens in the immunologic process.Minor DCs may intensively activate T cells,which may initiate the specific cellular immunity reaction. Their capacities of T cells activation are as 100-1000 folds as that of macrophages. They play an important role in the induction and regulation of immunologic response. It is confirmed by recent studies that arterial wall may offer a good microenviroment to the immunological reactions.A Vascular-associated lymphoid tissue（VALT）that similar to mucous membrane-associated lymphoid tissue in respiratory tract and gastrointestinal tract,exist in the normal endarterium.Some cell masses which are comprised of immunological competent cells and antigen presenting cells distribute diffusedly in these tissues.They may monitor and screen the potential endogeneous or exogenous harmful antigens in the vascular tissues.Vascular dendritic cells（VDCs） can be found in VALT.Normally speaking,only a very small number of VDCs which in an immature form,can be found in the vessel tunica intima and tunica adventitia. Aggregation of VDCs in the affected regions increase and,they always aggregate with T cells and macrophages in the inflammatory infilitrated regions.People presume that DCs may play a role in initiating inflammatory and immunological reactions in the genesis and development of AS for that DCs co-emerge with T cells in the weak affected regions.Recent studies showed that oxidated LDL and nicotine may promote the advancement of AS by activating DCs-mediated acquired immunological reactions.This suggest that DCs may play a role in the pathogenesis of AS and that they may play an important role in the regulation of the triggering and amplification of inflammatory and immunological reactions in AS.Astragalus polysaccharids,one of the most important natural active ingredients in Astragalus,have the function of bi-directional regulation of blood sugar,promoting immunity,enhancing macrophage activity and promoting the transformation of lymphocyte,activating T cells,B cells and promoting the formation of antibodies in order to fight against various viral and bacterial diseases.In addition,the study found that APS play an important role in various diseases,for instance,APS have anti-tumor effect,but APS can not inhibit effectively the growth of tumor cells in vitro, suggesting that its anti-tumor effects of APS may be associated with immune effects. APS can reduce the incidence of type 1 diabetes of the non-obese mice significantly, and protect the the ultrastructure of pancreatic islets,restore the balance between Th1 cells and Th2 cells.APS can reduce NF-kB mRNA expression levels in the renal cortex of SD rats,enhance the expression of IkB mRNA,and can control the progress of diabetic nephropathy effectively.APS can inhibit metal-induced oxidative stress and renal ischemia-reperfusion injury,stimulate the immune regulation,inhibit glomerular mesangial cell proliferation and matrix synthesis,as well as reduced the expression ofβ1 integrin in mesangial cell.In relation with the research of cardiovascular disease,APS can reduce total cholesterol,apolipoprotein H, low-density lipoprotein level of healthy people effectively,as well as triglycerides levels in more than 50-year-old middle-aged healthy population.On the study of the hamster model of diabetic cardiomyopathy,APS exert the effect of anti-diabetic cardiomyopathy by inhibiting the chymotrypsin-AngⅡsystem.Besides,more studies have shown that astragalus polysaccharide increase the content of cAMP in platelets and inhibit platelet aggregation and thrombosis through the inhibition of platelet calmodulin and phosphodiesterase activity.Moreover,Astragalus Polysaccharides are one of the major components in Astragalus injection,and in the treatment of angina pectoris,Astragalus injection can reduce the number of angina attacks,extend the attack time interval,improve the clinical efficacy significantly. Above all,Astragalus injection intervening in the development of CHD may be related to the immune regulation function of APS.Considering that DCs are key in natural defense and acquired immune and closely related to the occurrence and development of CHD,so in the treatment of CHD,whether APS play a role in regulating immune function though exerting on DCs?After treatment with APS,how the DCs functional status in antigen-presenting, and whether affect secretion of the functional proteins and immunomodulatory molecules,and the gene expression of these proteins in DCs or not.Study on genetic changes of DCs is the key in resolving these problems.Our studies can mainly be divided into 3 portions.Peripheral blood mononuclear cell（PBMC）and serum abstracted from clinical cases are the objectives of our study.The gene expressions and functional changes of PBMC derived DCs after treatment with APS is the starting point in our study.Through immunofluorescence,flow cytometry,DNA microarray,RT-PCR and ELISA,we have focused on the relationship between the occurrence and development of AS and mature status,immune function and other gene expression differences of PBMC peripheral blood-derived DCs after APS treatment,and discussed deeply the status and the role of antigen-presenting cell function after treatment with APS in the treatment of AS.Aiming to provide an experimental evidence and target of immune therapy for the immune-mediated hypothesis in the development of AS,and review new theoretical platform and technical mean for the prevention and treatment of cardio-cerebrovascular disease,this study have the long-term economic value and important social significance.The results are as follows:1.DCs derived from peripheral blood in a mature state after APS treatment,have the ability to induce T cell proliferation.1.1 Culture and identification of DCsIn cultured 2 day,cells adhesion to the bottom of polystyrene bottle in the control group,part cells are suspended growth in medium in the experimental group, smaller,and no significant difference shape.In cultured 6 day,cells adhesion to the culture bottle bottom with round or oval in shape in control group;cells,showing suspended growth in medium,part of them aggregation in clusters,dendritic-like protrusionsin on cell surface in different quatity and shape,have the best growth status in LPS group,100mg/L group.Large number of vacuoles-like structure in the majority of the cytoplasm cell,collapsed cells,and a large number of atypical cell debris can be seen in 200mg/L APS group.In APS 100mg/L group,the phenotype of human peripheral blood PBMC derived DCs are CD1α⁺,CD80⁺,CD83^low,CD86⁺, HLA-DR⁺by flow cytometry analysis.1.2 Detection cytokine IL-12 in cultured supernatant.Because of a large number of apoptotic cells in 200mg/L,the control group,LPS group,50mg/L APS group,100mg/L APS have been chosed to detect the cytokine IL-12 in cell supernatant.In culture 6 days,the content of IL-12 have significantly statistical difference in 4 groups of cells cultured supernatant（P=0.000）;compared the content of IL-12 in cells cultured supernatant of LPS group,l00mg/L APS with the control group,50mg/L APS group,there have significantly statistical difference （P=0.000）,and the content of IL-12 in cells cultured supernatant in the fomer two groups significantly higher than in the latter two groups.1.3 The phenotype of peripheral blood PBMC derived DCs varied in each group.Because of a large number of apoptotic cells in 200mg/L,the control group,LPS group,50mg/L APS group,l00mg/L APS have been chosed to detect the phenotype of peripheral blood PBMC derived DCs varied in each group.There have statistical difference in CD1α,CD86 of DCs phenotype between l00mg/L APS group,LPS group and control group,50mg/L APS group（P＜0.05）,and the CD 1α,CD86 of DCs phenotype in l00mg/L APS group,LPS group Showed up-regulated expression;there have statistical difference in HLA-DR of DCs phenotype between LPS group and 50mg/L APS group（P＜0.05）,and the HLA-DR of DCs phenotype in 50mg/L APS group Showed up-regulated expression.1.4 Allogenic mixed lymphocytic reactions in PBMC derived DCs in each group. There have a significant difference ³H-TDR incorporation rate in 4 groups（P＜0.01）.At the proportion of 1:50,1:20 of DCs mixed with T lymphocytes,there have significantly statistical difference between l00mg/L APS group,LPS group and other two groups in the rate of H-TDR incorporation（P＜0.01）,and the rate of H-TDR incorporation in l00mg/L APS group,LPS group significantly higher than the other two groups;but there have no statistical difference between the control group and 50mg/L in the ³H-TDR incorporation rate（P＞0.05）2 Detection different gene expression of the peripheral blood PBMC derived DCs by gene chipThe result of gene chip detection show that,the expression gene of functional protein after APS treatment in peripheral blood DCs have seven genes increased markedly,they are CD36,IGSF6,IL27,INHA,LIPA,MAP4K3 and SOD2,and belong to antigen recognition receptors,cytokine,cell enzymes and cell signal transduction system.And 26 genes showed decreased expression,they are ADAM19, ADAR,BASP1,BTG1,CCR7,CD40LG,CST7,CXCL9,CXCR4,DCTN2,EBB, ISG15,ICAM1,IFI16,IL15,ISG20,CD207,LTA,MARCKS,MARCKSL1, NFKB1,NFKB2,PNRC1,RAC1,RELB and TNFRSF11B,and belong to cell enzymes,antigen recognition receptors,cytokine receptor,cytokine and cellular signal transduction systems.The peripheral blood PBMC after APS treatment are reduced in the expression of cell adhesion and chemotactic migration of DCs receptors and related proteins such as AD AM19,CCR7,CD40LG,CST7,CXCL9 and CXCR4;the gene expression with related to the induction of cell proliferation and activation signal transduction pathway and functional protein are also decreased, such as ADAR,BASP1,BTG1,DCTN2,ISG15,ISG20,MARCKS,MARCKSL1, NFKB1,NFKB2,PNRC1,RAC1,RELB and TNFRSF11B;and the gene expression with related to antigen-presenting and immune response related protein are decreased, such as EBI3,IFI16,IL15,CD207,and LTA.In upregulated expression genes,there have antigen-binding protein gene such as CD36,digestion and degradation of antigen related cell and lysosome protein gene such as LIPA and SOD2,as well as inhibition inflammation and immune responses protein gene expression such as IL27 and INHA.3 Detection the results of gene chip by fluorescence quantitative PCR showed that gene chip have better credibility in detection DCs gene.Application fluorescence quantitative PCR in detection of microarray gene expression of CD36 and IL-27,as well as down-regulated expression gene FIF16 showed that,compared with control group,CD36 and IL-27 gene expression in DCs group after APS treatment increased width,FIF16 gene expression decreased width is similar with gene chip detection.Through above three-part experiment,we can draw the conclusions as follows:①The phenotype expression in peripheral blood DCs after APS treatment are in a mature state; ②The levels IL-12 secreted in peripheral blood PBMC derived DCs after APS treatment,are significantly higher than the control group,and have the ability to induce allogenic lymphocytic proliferation;③The expression gene of functional protein after APS treatment in peripheral blood DCs have seven genes increased markedly,they are CD36,IGSF6,IL27,INHA, LIPA,MAP4K3 and SOD2,and belong to antigen recognition receptors,cytokine, cell enzymes and cell signal transduction system.And 26 genes showed significant decreased expression,they are ADAM19,ADAR,BASP1,BTG1,CCR7,CD40LG, CST7,CXCL9,CXCR4,DCTN2,EBI3,ISG15,ICAM1,IFI16,IL15,ISG20,CD207, LTA,MARCKS,MARCKSL1,NFKB1,NFKB2,PNRC1,RAC1,RELB and TNFRSF11B,and belong to cell enzymes,antigen recognition receptors,cytokine receptor,cytokine and cellular signal transduction systems.④Detection the CD36 and IL-27 gene which upregulated expression,and IFI16 gene which downregulated expression in microarray by fluorescence quantitative PCR,verified the results of the gene microarray.更多还原