摘要
冠心病(Coronary Heart Disease,CHD)是人类健康的主要杀手。心脏病流行病学调查资料表明,随着生活方式的改变,我国的CHD患病率和死亡率呈逐年上升趋势,据世界卫生组织(WHO)估计：到2020年左右,我国会迎来冠心病的“流行”顶峰。动脉粥样硬化(Atherosclerosis,AS)是冠心病发病的病理基础,人们从500年前埃及木乃伊的动脉中就已发现粥样硬化性病变。人类认识AS是一种疾病并对其发病机制进行相关研究已有100余年的历史。有关AS的发病机制学说数量众多,其中包括内皮损伤学说、脂质浸润学说、血栓源性学说、动脉平滑肌细胞增殖学说等。而自1999年Ross提出AS的炎症反应机制以来,AS的炎症反应机制也已经广泛被医学界认可。研究证实,炎症参与了AS形成及发展的各个阶段,直至最终的血栓并发症。而近年来,免疫因素在AS的发生发展中的作用正在被人们认识并引起重视,更有学者提出AS是一种自身免疫性疾病。在AS中,多种参与免疫细胞和炎症因子构成了错综复杂的相互协同、相互影响的网络,炎症和免疫共同影响着AS的进程。
T淋巴细胞是重要的免疫细胞,参与特异性免疫反应,它与AS的关系国际上已有不少研究。AS的病理改变特点为平滑肌细胞增殖、脂质沉积、巨噬细胞和淋巴细胞浸润、泡沫细胞形成。在人类AS的各个时期都有不同程度的淋巴细胞浸润,其中以T细胞为主。活动性斑块与稳定性斑块比较,T细胞显著增加。许多关于人类和啮齿目动物的研究证实,T淋巴细胞促进粥样硬化斑块的炎症反应,影响损害的进展及重塑。主要表现在以下几点：1.T细胞与斑块中其他免疫细胞及分子相互作用,促进粥样硬化；2.T细胞参与ox-LDL抗体、Hsp抗体等与AS相关的自身抗体形成；3.杀伤性T细胞溶解破坏巨噬细胞,释放大量促粥样硬化物质；4.T细胞在不稳定性斑块形成及斑块破裂中可能起一定作用。
T细胞活化是其发挥病理生理作用的前提。而T淋巴细胞的活化需要两个信号,第一个信号是T淋巴细胞表面的TCR-CD3复合物同抗原递呈细胞(APC)上的MHC-Ⅱ类抗原肽复合物结合,赋予免疫应答的特异性,但不引起T细胞增生和分泌细胞因子；第二个信号是抗原非特异性的共刺激信号,由T细胞上的共刺激分子与APC表面的配体结合所诱导,启动维持并调节活化级联反应,决定了T细胞是活化增殖,或者转变为无反应状态甚至凋亡。重要的共刺激分子及其配基有：B7/CD28,血管细胞黏附分子(VCAM)-1/迟现抗原(VLA)-4,细胞间黏附分子(ICAM)-1/淋巴细胞功能相关抗原(LFA)-1, LFA-3/CD2, CD40/CD40配基,CD70/CD27等。
近年来,一些新的共刺激分子陆续被发现,它们似乎在调节已活化的T细胞反应中有独特的重要性。负性共刺激分子PD-1 (Programmed death-1)和其配体PD-L (Programmed death-1 ligand)相互作用提供淋巴细胞活化的抑制信号,和正性共刺激信号形成了一个复杂的信号网络,综合调节免疫反应,在免疫活化和耐受中起关键作用。
PD-1分子是免疫球蛋白超家族激活诱导的抑制性受体,是拥有免疫受体酪氨酸抑制基序(ITIM)和免疫受体酪氨酸转换基序(ITSM)的Ⅰ型跨膜蛋白,可诱导性表达于活化的CD4+T细胞和CD8+T细胞、B细胞、NK细胞及骨髓细胞。PD-L1(B7-H1)和PD-L2(B7-DC)是PD-1的两个配体,PD-L1主要表达在树突状细胞、单核/巨噬细胞、B细胞、激活的T细胞,在内皮细胞、肿瘤细胞上也有表达,IFN-α、β、γ可上调其表达。PD-L2在树突状细胞、单核细胞上表达,IL-4、GM-CSF在体外强力诱导树突细胞上PD-L2的表达。PD-1-PD-L结合后,发挥对效应T细胞的负性调控作用,可抑制T细胞增殖和产生细胞因子IL-10、IFN-γ等。在免疫反应的起始部位和效应部位双重抑制免疫反应,防止过度的免疫损伤和自身免疫性疾病的发生,有利于维持机体的免疫自稳。
PD-1负性共刺激信号的缺失会导致多种自身免疫性病变。动物实验显示,PD-1信号缺失导致小鼠发生类风湿关节炎及狼疮样自身免疫性肾炎,导致NOD小鼠自身免疫性糖尿病的快速进展。增加PD-1负性共刺激信号可使AS兔粥样斑块中T细胞浸润减少及炎性反应减轻,减轻实验性脑脊髓膜炎(EAE)程度,使脊髓浸润的巨噬细胞、CD4+、CD8+T细胞消失。近年来研究者们对PD-1在临床疾病的表达的研究发现,病毒感染(如HBV、HIV)、寄生虫感染、移植免疫、自身免疫病(如系统性红斑狼疮、系统性硬化)、肿瘤等多种疾病,都存在PD-1及其配体PD-L的表达异常,显示出PD-1信号在对维持外周耐受及免疫自稳方面起到保护性作用。
他汀作为羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,是有效的降脂药物。除调脂作用之外,他汀还具有包括改善内皮功能、增加NO生物利用率、抗氧化、稳定斑块、调节祖细胞、抗炎、免疫调节等多重血管保护作用。其抗炎及免疫调节方面的功效已在多种疾病如类风湿关节炎、多发性硬化的应用中体现。
AS是一个慢性炎症反应,且具有自身免疫的特点,而PD-1在其中的作用机理尚不清楚。本研究通过对免疫负性调控信号分子PD-1在CHD患者外周血T淋巴细胞(PBTC)的表达情况的检测,初步分析了其在AS发病及进展中的意义；利用特异性PD-1阻断抗体研究其对AS患者PBTC的免疫学调节作用,进一步推测PD-1在AS中的调控机制；并通过观察辛伐他汀(Sim)对PD-1在CHD患者PBTC上的表达的调节作用,为PD-1作为AS免疫学治疗提供新的理论依据。
第一部分：CHD患者PBTC上PD-1的表达
目的：研究PD-1在CHD患者PBTC上的表达,初步探讨其在AS发生发展中的意义。
方法：选择CHD患者(55例)为研究对象,其中稳定性心绞痛(SA)患者27例,急性冠脉综合征(ACS)患者28例,30例经冠脉造影证实的非CHD患者为正常对照组,密度梯度离心法分离外周血单个核细胞(PBMC),分别用免疫荧光、流式细胞术及RT-PCR检测PD-1蛋白及mRNA在T淋巴细胞上的表达情况。
结果：
1.直接免疫荧光观察PD-1蛋白表达于PBTC的表面。
2.流式细胞术检测PD-1蛋白表达PD-1在PBTC上的表达在SA患者组表达率为(9.29±2.34)%,ACS患者组为(8.19±2.36)%,正常对照组为(18.52±3.28)%。两组CHD患者的PD-1表达率低于正常对照组,差异具有显著性P<0.001)。
3.RT-PCR检测]PD-1mRNA在PBTC的表达PD-1mRNA结果显示SA组患者表达量为0.552±0.079,ACS组为0.477±0.081,均低于正常对照组的0.928±0.057(P<0.001)。
结论：PD-1在CHD患者PBTC上的表达明显降低,这可能在AS的发生及发展中起到作用。
第二部分PD-1对体外培养CHD患者PBTC的免疫学调控作用
目的：研究PD-1对体外培养CHD患者PBTC的免疫学调控作用。
方法：PBTC分离与纯化同前。7例CHD患者和正常对照来自前一实验样本人群。正常人及CHD患者的PBTC新鲜分离纯化后,各分为4组,与另一健康人来源的成熟树突状细胞(mDC)以20：1比例混合,置于37℃的CO2培养箱共培养,72小时后收集各组上清液ELISA法检测IFN-γ、IL-4。3H-胸腺嘧啶核苷(3H-TdR)掺入率反映T淋巴细胞的DNA合成。
实验分组：1)PBTC组：PBTC加等体积的PBS作为阴性对照组；2)PBTC+mDC组：PBTC与mDC共培养；3)PBTC+mDC+PD-1Ig组：PBTC与mDC共培养体系中加入鼠抗人PD-1抗体(终浓度为10μg/ml)；4)PBTC+mDC+Ig组：PBTC与mDC共培养体系中加入鼠抗人同型对照Ig(终浓度为10μg/ml)。
结果：
1.PD-1共刺激分子对CHD患者和正常人PBTC增殖作用的影响：正常对照及CHD患者不同处理组之间差异有显著性意义(P<0.001)。PBTC+mDC+PD-1Ig组3H-TdR掺入率较PBTC+mDC组增加有显著性意义(P<0.001),而加入同型对照抗体则无变化(P>0.05)。析因方差分析显示,CHD患者组PD-1阻断后,PBTC增殖程度低于对照组,即PD-1分子抑制T细胞增殖的作用减弱。
2.PD-1共刺激分子对CHD患者和正常人PBTC分泌细胞因子作用的影响：正常对照及CHD患者PBTC组、PBTC+mDC组、PBTC+mDC+PD-1Ig组和PBTC+mDC+Ig组的IFN-γ、IL-4表达量组间差异具有显著性。LSD比较分析表明,PBTC+mDC组比PBTC组IFN-γ和IL-4分泌增加有显著性意义(均P<0.001)；在PBTC+mDC组加入PD-1-Ig后,IFN-γ和IL-4分泌均增加,有显著性意义(均P<0.001),而加入同型对照抗体则变化无显著性意义(P>0.05)。析因方差分析显示,CHD患者组PD-1阻断后,细胞因子产生增加程度低于对照组,即PD-1抑制T细胞因子产生的作用减弱。
结论：CHD患者及正常人体内PD-1共刺激分子在抑制PBTC增殖与分泌IFN-γ和IL-4发挥着重要的作用。CHD患者较正常人比较这种抑制作用相对减弱。
第三部分辛伐他汀对PD-1在CHD患者PBTC表达的调节作用
目的：观察辛伐他汀(Sim)对PD-1在CHD患者PBTC表达的调节作用,初步探讨他汀药物的免疫学调节AS的新机制。
方法：取12例CHD患者新鲜外周血15ml,密度梯度离心法得到PBMC,并分为3个组：取分离出的CHD患者PBMC,同一患者细胞再分为空白对照组与Sim组：分别加入等体积的PBS与Sim(终浓度分别为10-6mol/L和10-5mol/L)。24小时小时后收集细胞,洗涤并调整浓度为5×105/ml。流式细胞术检测PD-1在PBTC的表达。
结果：流式细胞术检测PBTC上PD-1蛋白的表达CHD患者PBMC在Sim作用24小时后,PBTC上PD-1的表达上调,10-5 mol/L Sim组较对照组及10-6 mol/LSim组诱导PD-1表达是上调明显,差异有显著性(P<0.001)。
结论：他汀具有上调PD-1在CHD患者PBTC表达的作用,这一作用可能参与了其对CHD的免疫学调控机制。
Coronary Heart Disease (CHD) is the main killer of human health. Heart disease epidemiology data indicates that with the changes of lifestyle, CHD in China has gradually increasing morbidity and mortality. The World Health Organization (WHO) estimates:around 2020, China will face the epidemic peak of CHD. Atherosclerosis (AS) is the pathologic basis of cardiac-cerebro vascular disease.500 years ago, atherosclerotic lesions had been found in the arteries of Egyptian mummies. It has been more than 100 years of history for human to understand AS and study its pathogenesis. There are different theories to explain the pathogenesis of AS, including endothelial injury, lipid infiltration, thrombosis and arterial smooth muscle cell proliferation. AS was categorized to inflammatory diseases by Russell Ross in 1999, Ross proposed AS is the arterial wall endothelial cells and smooth muscle cells after injury by an inflammatory fibroid tissue reaction. Recent advances in basic science have established a fundamental role for inflammation in mediating all stages of this disease from initiation through progression, ultimately, the thrombotic complications of AS. Now, there is growing evidence that AS is a chronic inflammatory disease, autoimmunity in AS may play an important role.
T lymphocytes (T cells) are important immune cells involved in immune response specificity. Current evidence supports a central role for T lymphocytes in all phases of the AS process.
AS is a complex disease characterized by smooth muscle cell proliferation, cholesterol deposition, infiltration of macrophages and lymphocytes and foam cells formation. It is confirmed that at all stages of atherosclerosis there are different degrees of lymphocyte infiltration, in which T cells were predominant. Compared with stable plaques, T cells were significantly increased in unstable plaques. Evidence from many human and rodent studies has established that T lymphocytes enhance inflammation in atherosclerotic plaques and contribute to lesion progression and remodeling. T cells interact with other components in plaque, and promote atherosclerosis; T cells involved in atherosclerosis-related autoantibodies formation such as ox-LDL antibodies, Hsp antibodies; Natural killer T cell dissoved macrophages and released of a large number of substances to promote atherosclerosis; T cells contribute to atherosclerotic plaque instability.
The precondition for playing its pathophysiological role is T cell activation. The T lymphocyte activation requires two signals. The first signal is the combine of TCR-CD3 on T cell surface with MHC-Ⅱantigen peptide on antigen presenting cells (APC) Giving the specificity of immune response, but not T cell proliferation and secretion of cytokines. The second signal is co-stimulation signal, the T cell costimulatory molecule on the surface of APC induced by ligand combnding, starts, maintains and regulates the activation cascade, determines the T cells to proliferate, respond to state and even into apoptosis. Important costimulatory molecule and its ligand are:B7/CD28, VCAM-1/VLA-4, ICAM-1, LFA-3/CD2, CD40/CD40L, CD70/CD27, etc.
CD28/CTLA-4 (cytotoxic T lymphocyte-associated antigen 4)-B7-1/B7-2 and CD40-CD40L signaling pathways has been shown to play an important role in regulating T cell activation and immune tolerance. In recent years, some new B7/CD28 superfamily were successively found, they seem to have had unique importance on regulation of activated T cell response. Like CTLA-4/B7, PD-1/PD-L (Programmed death-1/Programmed death-1 ligand) interaction also provides inhibitory signals of lymphocyte activation. And the complex network composed by positive and negative costimulation signals plays a critical role in immune activation and tolerance.
PD-1 molecule is the immunoglobulin superfamily inhibitory receptor, is a type 1 transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), which is induced upon the activation of CD4 and CD8 Tcells as well as B, NKT and myeloid cells. PD-L1 (B7-H1) and PD-L2 (B7-DC) are the two ligands of PD-1, PD-L1 is expressed mainly in dendritic cells, monocytes/macrophages, B cells, activated T cells, as well as endothelial cells, tumor cells, IFN-α,β,γcan induce its expression. PD-L2 is expressed on dendritic cells, monocytes, IL-4. GM-CSF in vitro strongly induced the expression of PD-L2 on dendritic cells. PD-1 delivers a negative costimulatory signal to effector T cells by engagement with its ligands, inhibits T cell proliferation and cytokine production, inhibits immune response at initial and effect stages to prevent excessive immune injury and autoimmune diseases, maintains immune homeostasis.
PD-1 negative costimulatory signal deficiency will lead to a variety of autoimmune diseases. Animal experiments showed that PD-1 signal deficient could develop lupus-like glomerulonephritis and arthritis in mice, and leading to autoimmune diabetes with rapid progress in NOD mice. Increasing in PD-1 negative costimulatory signals decreased T cells infiltration and inflammatory reaction in AS rabbit plaque; relieved experimental autoimmune encephalitis and infiltration of macrophages, CD4+, CD8+T cells in spinal cord. In recent years, researchers have found that the expression of PD-1 and its ligands were abnormal in many clinical diseases:viral infections (such as HBV, HIV), parasitic infections, transplantation immunology, autoimmune disease (such as SLE, systemic sclerosis), cancer and other disease, showing that PD-1 signaling played a protective role in the maintenance of peripheral tolerance and immune homeostasis.
Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are potent cholesterol-lowering drugs. In addition to their cholesterol-lowering properties, statins exert a number of so-called'pleiotropic', vasculoprotective actions that include improvement of endothelial function, increased nitric oxide (NO) bioavailability, antioxidant properties, stabilisation of atherosclerotic plaques, regulation of progenitor cells, inhibition of inflammatory responses and immunomodulatory actions. Anti-inflammatory and immunomodulatory effects of statins were demonstrated in various of diseases such as rheumatoid arthritis, multiple sclerosis.
AS is a chronic inflammatory disease, and has the characteristics of autoimmune, in which the mechanism of PD-1 is unclear. In this study we had investigate the negative costimulatory molecule PD-1 expression on PBTC in CHD patients, a preliminary analysis of its pathogenesis in AS was conducted. We had used of specific blocking antibodies to speculated the mechanism of PD-1 in AS. At last, we observed the effect of Simvatatin (Sim) on the expression of PD-1, which might provide a new evidence of Sim in immunological therapy of AS.
PartⅠ:PD-1 expression on PBTC in CHD patients
Objective:To investigate the expression of PD-1 on PBTC in CHD patients.
Methods:55 patients with CHD were enrolled into our research at the Department of Cardiology of our hospital.30 patients without CHD confirmed by coronary angiography were served as controls.The PBTCs of CHD patients and controls were isolated by Ficoll density gradient medium and nylon wool column. The expression of PD-1 was detected by direct immunofluorescence, flow cytometry and half-quantitative RT-PCR.
Results:PD-1 expressed on PBTC of CHD patients and controls.The expression level of PD-1 in stable angina(SA) group and acute coronary syndrome(ACS) group was found to be lower than that of the normal control group (P<0.001, P<0.001, respectively). But there was no statistically significant difference between SA group and ACS group (P>0.05).
Conclusions:The expression of PD-1 on PBTC from patients with AS was significantly down-regulated, which might play some role in the pathogenesis of CHD.
Part 2 Immunological regulation of PD-1 on PBTC of CHD patients cultured in vitro
Objective:To detect the Immunological regulation of PD-1 on T lymphocyte of CHD patients cultured in vitro.
Methods:PBTC separation and purification as former.7 CHD patients and controls were selected from the population involved in our study. PBTC were fresh purified from patients and controls, and mixed with purified mDC from a third healthy individual at ratio of 20:1. Blocking antibodies were added to some cultures at a concentration of 10μg/ml, and they were instead by equal volume of PBS in the PBTCs alone group, or isotype antibodies in the control group. Then cultures were divided into 4 groups:PBTC group, PBTC+mDC group, PBTC+mDC+PD-1-Ig group, and PBTC+mDC+Ig group. Cells were cultured in 24 well plates for 72 hours in CO2 incubators at a temperature of 37℃.3H-thymidine (3H-TdR) incorporation was measured to evaluate DNA synthesis of T lymphocytes. The supernatant of each group was collected for cytokine detection.
Results:
1. The effect of PD-1 on proliferation of PBTC:The difference between groups in both controls and CHD patients was significant (P<0.001).3H-TdR incorporation increased more in PBTC+mDC+PD-1Ig groups than PBTC+mDC groups (P <0.001). Changes in PBTC proliferation caused by PD-1 blocking were less in CHD patients
2. The effect of PD-1 on production of cytokines by PBTC:Differences between groups on production of IFN-γand IL-4 were significant (P<0.001). LSD test showed that, IFN-γand IL-4 production was significantly induced in PBTC+mDC group than in the PBTC group (P<0.001). Adding PD-1-Ig into PBTC+mDC group, IFN-γand IL-4 production was increased more significantly (all P<0.001). Changes in cytokine production caused by PD-1 blocking were less in CHD patients.
Conclusions:PD-1 plays a critical role in inhibition of PBTC proliferation and production of cytokine, both in cantrols and CHD patients. But its capacity of inhibition seemed weakened in CHD patients.
Part 3:Regulation of Sim on the expression of PD-1 on PBTC from CHD patients
Objective:To observe the effects of Sim on the expression of PD-1 on PBTC from CHD patients, find out new mechanisms of statins on the immune regulation on AS.
Methods:15ml fresh peripheral blood of 12 patients with CHD was drawn for PBMC purification. PBMC from each patient were divided into three groups:control group (equal volume of PBS were added) and two Sim groups (final concentrations of 10-6 mol/L,10-5 mol/L).24 hours later, cells were collected, washed and adjust the concentration of 5×105/ml. PD-1 expression on T cells was detected by flow cytometry.
Results:PD-1expression on T cells was significantly up-regulated by Sim with a concentration of 10-5 mol/L in patients with CHD (P<0.001).
Conclusions:Statins have capacity of up-regulation of PD-1 expression on peripheral blood T cells of patients with CHD, Which may be involved in its immunomodulatory action in CHD.
引文
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