【作者】 周音频；

【导师】 黄岚；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 研究背景冠心病的病理基础是动脉粥样硬化,已经证实动脉粥样硬化是一种慢性反应性炎症疾病过程。动脉粥样硬化斑块在形成和进展中,其性质和特点有很大差异。除斑块体积对血管腔血流有直接影响外,斑块本身的成分和性质对患者临床表现及预后影响更大。含有较大坏死性脂核、纤维帽薄、炎症细胞浸润多的斑块被称为易损性斑块(vulnerable plaque),这类斑块炎症反应程度重,最容易破裂并导致血栓形成,使血流急剧减少或者完全阻断,是临床发生急性冠状动脉综合征的主要发病机制。逆转易损斑块、促进其消退及/或干预易损斑块使其向稳定型斑块转化,是防治急性冠脉综合征的重要措施,也是临床上目前需要急待解决的关键问题。加强对易损性斑块中炎症过程启动和强化机制的研究,对促进易损性斑块稳定并更好地防治急性冠脉综合征等高危人群,具有显著的经济和社会效益。在动脉粥样硬化形成过程中,免疫系统和免疫细胞起到了重要作用。作为动脉粥样硬化主要危险因素的高脂血症和高血糖等因素可以引起免疫应答的启动,已经证实与动脉粥样硬化病变关系密切的抗原有氧化修饰的低密度脂蛋白(Ox-LDL)、热休克蛋白(Hsp)和β2-糖蛋白Ⅰ(β2-glycoproteinⅠ)。作为主要炎症细胞之一的T淋巴细胞,表现对动脉粥样硬化相关抗原的应答反应,促进动脉粥样硬化的发生和进展。在动脉粥样硬化的形成和演变中不同的T淋巴细胞亚群起不同的作用,其中对动脉粥样硬化发生起优势作用的T细胞类型是CD4阳性T辅助细胞(Th)。临床上急性冠脉综合征的冠脉病理病变以易损性斑块为主,对斑块性质研究发现易损性斑块炎症程度重,无论是斑块局部还是全身血循环Th1和Th2比例均存在不平衡,这导致粥样斑块的炎症加重,使斑块向不稳定趋势进展。程序性细胞死亡因子1(Programmed cell death 1,PD-1)是近年来发现的新的介导负调控信号的共刺激分子受体,在激活的T细胞、B细胞和一些髓样细胞上表达。PD-1属于CD28家族,其配体PD-Ls属于共刺激分子B7家族,主要有PD-L1(B7-H1)和PD-L2(B7-DC)。PD-L1和PD-L2在巨噬细胞和DCs上均有表达,长期慢性病毒感染可以使T细胞PD-1表达持续上调,同时处于功能衰竭状态;而用抗体阻断PD-1则可以使衰竭的T细胞增殖和分泌细胞因子水平的功能均恢复正常。已经有研究发现CD4阳性T淋巴细胞在粥样硬化斑块中表达,其中PD-1表达情况如何尚了解甚少,对PD-1进行阻断性干预,对形成粥样硬化斑块模型体内T淋巴细胞功能以及对斑块易损性的影响,也不清楚。本实验用免疫组化方法检测粥样斑块形成中PD-1的表达水平,然后用抗PD-1抗体干预,评价CD4阳性细胞功能的变化和对粥样斑块性质的影响。研究方法本研究分三部分,第一部分:选取不同类型的冠心病人55例(不稳定型心绞痛31例、稳定型心绞痛24例),用ELISA方法检测血清ox-LDL及外周血PBMC释放细胞因子IFN-γ和IL-4水平,同时用流式细胞法检测外周血CD4~+和CD4~+PD-1~+双阳性细胞比例,并与正常的对照者进行比较,探讨PD-1在CD4阳性T淋巴细胞上的表达与冠心病炎症程度的关系;第二部分:取C57BL/6J ApoE基因敲除鼠及相同遗传背景野生型小鼠,喂食高脂饲料,21周后处死小鼠,石蜡包埋血管后作连续切片,行HE染色及PD-1免疫组化染色,检测斑块面积和PD-1表达情况;用辛伐他汀干预以后,观察斑块面积和PD-1表达水平的变化;第三部分:制成动脉粥样硬化动物模型,然后用抗PD-1抗体干预实验动物,观察其粥样斑块性质的改变,并同时测定在体分离的CD4阳性细胞增殖和分泌细胞因子的能力,探讨PD-1/PD-Ls信号途径对斑块易损性的影响及可能机制。结果1.外周血中分离PBMC培养后,其上清中的IFN-γ水平以UA组中最高,超过SA组和对照组,差异有显著性(P＜0.05),SA组IFN-γ水平也较对照组高;三组的IL-4值比较则差异不是很明显;UA组血清中ox-LDL值超过SA组(P＜0.05),对照组中ox-LDL值最低;2.SA组和UA组外周血PBMC中CD4~+细胞比例均较对照组增高(P＜0.05),SA组和UA组外周血PBMC中CD4~+PD-1~+双阳性细胞比例均较对照组增高(P＜0.05),其中UA组较SA组增高更明显差异有显著性(P＜0.05),提示随炎症程度增高,CD4阳性细胞上PD-1的表达增强;3.AS模型组可见动脉粥样硬化斑块明显形成,斑块面积较对照组明显增大,其粥样斑块表达PD-1水平也增高;经辛伐他汀治疗组,斑块面积明显减小,PD-1表达也减弱;4.抗PD-1抗体干预组和AS组均可见血管腔有明显动脉粥样硬化斑块形成,经计算二组斑块面积无显著性差别。但抗体干预组斑块中有含脂质和坏死细胞的核心,表面纤维帽薄,斑块中含胶原的量偏少,平均光密度值小,与AS组比较有显著性差异(P＜0.05);5.抗体干预组的斑块中,免疫荧光检测有较多的CD4阳性细胞浸润,抗体干预组CD4阳性细胞平均光密度值较高,与AS组比较有显著性差异(P＜0.05);6.抗体干预组脾脏分离的CD4阳性细胞~3H-TdR掺入量明显较AS组增高,差别具有统计学意义(P＜0.05),提示细胞增殖能力增强;抗体干预组CD4阳性细胞分泌IFN-γ和TNF-α水平明显较AS组高,与AS组比较有显著性差异(P＜0.05)。结论1.在UA患者体内,外周血PBMC分泌细胞因子的能力明显增强,CD4阳性细胞激活的比例也较SA高,提示其炎症程度重,ox-LDL作为刺激免疫应答的抗原之一,可能是炎症激活的原因。2.冠心病患者中,随炎症程度增高,CD4~+细胞和CD4~+PD-1~+双阳性细胞比例均增高,作为负性调控因子的PD-1表达增强,其作用可能是反馈性抑制CD4~+细胞的过度激活。3.在ApoE基因敲除小鼠,随动脉粥样硬化斑块形成程度加重,PD-1在斑块的表达增加,经辛伐他汀干预后斑块体积减小,PD-1的表达减少,进一步提示其限制和下调炎症的作用。4.给予抗PD-1抗体后,斑块内胶原减少和CD4阳性细胞浸润增加,表现易损性增加改变,进一步证实PD-1/PD-Ls信号途径在斑块演变中具有下调炎症的作用。5.经抗PD-1抗体干预后,同时分离的CD4阳性T淋巴细胞表现增殖能力增强和分泌细胞因子量增加,提示PD-1的负性调节作用是通过抑制炎症细胞功能实现的。更多还原

【Abstract】 Background and ObjectivesThe pathogenesis of coronary heart disease（CAD） is atherosclerosis（AS）.There are strong evidences that atherosclerosis is a chronic inflammation disease.The nature and feature of atherosclerosis are different markedly during its development and progression. Besides blood vessel narrowing,the composition and characteristic of plaque can exert great effects on clinical manifestation and prognosis of CAD patients.The vulnerable plaque, characterized by a big necrotic lipid nuclus,thin fibrous cap and abundant inflammatory cells infiltration,once rupture and thrombosis,it can further reduce blood flow or lead to coronary artery blockade.Vulerable plaque is a major factor contributing to acute coronary syndrome（ACS）,reversal and stabilization of vulerble plaque is a important therapeutic target in ACS clinical strategies.Facilitating the study associated with starting and reinforcement of inflammation process in plaque has great economic and social profits. During the process of atherosclerosis development,immune system and immunocytes play key roles.As major risk factors,hyperlipemia and hyperglycemia can induce immune response in vessel wall.Some researches have provided evidences that oxidated low-density lipoprotein（ox-LDL）,heat shock protein（Hsp） andβ2-glycoprotein I have been regarded as antigens related to atherosclerosis.During the development and progression of atherosclerosis,the effects of different subgroups of T lymphocyte are variant,CD4⁺ helper T cell has a dominant effect on development of atherosclerosis.The artery lesions of ACS are composed of rich vulnerable plaques.As Th1 over differentiation, imbalance of Th1 and Th2 in local lesions and circulation results in inflammation aggravation,lead to plaque destabilization.As a recent discovered negative costimulatory molecule,programmed cell death 1（PD-1） is a receptor expressed on activated T cell,B cell and myeloid cell.It belongs to CD28 family,its ligands（PD-Ls） such as PD-L1 and PD-L2 belong to B7 superfamily,expressed on macrophage and dendritic cell.In chronic viral infection PD-1 has been upregulated on T cell,but whether or not PD-1 expressed on CD4⁺ T cell in atherosclerosis has little been known.Our hypothesis is:If PD-1 expression on T cell in atherosclerosis be blocked,the T cell function and plaque vulnerability might alter. In this study the expression of PD-1 in atherosclerosis was immunohistochemically investigated,then CD4⁺ T cell function was mesured and plaque composition was tested using monoclonal antibodies against PD-1.MethodsThe first part:56 CAD patients were included in the study,ustable angina（UA） 30 and stable angina（SA） 26 cases.Serum ox-LDL level,cutured supernatant interferon-γ（IFN-γ） and interleukin-4（IL-4） of peripheral blood monouclear cell（PBMC） were detected by enzyme linked immunosorbent assay（ELISA）.Flow cytometry was used to detect the expression of CD4⁺ and CD4⁺PD-1⁺ on PBMC in peripheral blood,and compared with control group,to investigate the relationship between the expression of PD-1 on CD4+ T cell and the inflammation degree in CAD patient.The second part:Twenty C57BL/6J ApoE gene knock out(ApoE^-/-) mice and 8 wild type C57BL/6J mice were fed with high fat diet.Twenty-one weeks later,all the mice were executed for the collection of aortas which were dissected from aortic root to abdominal aorta.Then the aortic sections were imbedded with paraffin for HE staining and PD-1 immunohistochemistry,the plaque area and expression of PD-1 were mesured.Interfered with Simvastatin,the variation of plaque area and expression of PD-1 were observed.The third part:The animal models of AS were established,and one of the AS groups was interfered with anti-PD-1 antibody,then the atherosclerotic morphology was studied, proliferation capability and excreted cytokine of CD4⁺ T cell were mesured.The vulnerability of atherosclerotic plaque and PD-1/PD-Ls signal pathway were investigated in order to reveal the latent mechanism.Results1.The PBMC cultured supernatant IFN-γand IL-4 levels in peripheral blood were increased obviously in CAD patients compared with control group（P＜0.05）,IFN-γand IL-4 levels in UA group were further higher than those in SA group（P＜0.05）,serum ox-LDL had a similar result.The levels of IL-4 had no significant difference in three groups.2.The percentage of CD4⁺ T cell of PBMC was higher in CAD patients than that in control cases（P＜0.05）.The extents of CD4⁺PD-1⁺ double positive expression on PBMC were both higher in UA and SA groups than that in control group（P＜0.05）,it increased markedly in UA（compared with SA P＜0.05）.3.There were significant atherosclerotic plaques in AS model,plaque area of AS group was more remarkable than that of contol group.There was more expression of PD-1 on plaque in AS group than that in control group（P＜0.05）,the plaque area and expression of PD-1 reduced in simvastatin group.4.The plaque area was not different significantly in anti-PD-1 intervention group and AS group,but the atherosclerotic plaque was characterized with more lipid,a great necrotic core,thin fibrous cap and fewer collagen.The average optical density（AOD） of collagen in anti-PD-1 intervention group was fewer compared with AS group（P＜0.05）.5.There were more CD4⁺ T cells infiltrated in atherosclerotic plaque in anti-PD-1 intervention group when examined by immunofluorescence method.The AOD value of CD4⁺ T cell infiltration in anti-PD-1 intervention group was higher compared with AS group(（P＜0.05）.6.Cultured for 72 hours,CD4⁺ T cells separated from spleen had more ³H-TdR incorporation,higher IFN-γand IL-4 levels in anti-PD-1 antibody intervention group compared with AS group（P＜0.05,respectively）.These results indicated the CD4⁺T cell had increased capability to proliferation and secreting cytokines.Conclusions1.In UA patients,as a antigen associated with atherosclerosis,ox-LDL may activate CD4+ T cells to secret more cytokines,lead to increased inflammation.2.The expression of PD-1 on CD4⁺ T cell increase when inflammatory response is enhanced,indicate the inhibitory effect of PD-1 on CD4⁺ T cell activation.3.The expression of PD-1 increase on atherosclerotic plaque during development of atheroma in ApoE-/- mice.Simvastatin can decrease plaque area and expression of PD-1 reduced,indicated the role of PD-1 may be limiting and downregulating inflammation. 4.Anti-PD-1 antibody intervention can increase CD4+ T cell infiltration and decrease collagen composition in plaque,lead to progression of vulnerable plaque.5.Anti-PD-1 antibody intervention can enhance the proliferation capacity and increase the secreted cytokines of CD4⁺ T cell,indicated the effect of PD-1/PD-Ls signal pathway on down-regulation inflammation.更多还原