T淋巴细胞单克隆增殖与急性冠脉综合症发生发展关系的研究
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摘要
AS是冠心病、脑血管意外和外周血管疾病的主要病理基础。近百年来,其发病率直线增高、发病范围覆盖越来越广、发病年龄也逐渐年轻化,已经成为危害人类健康的“第一杀手”。为了阐明AS的发生机制而最终征服AS,医学工作者在病理学、流行病学和病因学方面展开了一系列研究。然而,AS的发生发展机制仍未明确。
纵观AS的研究过程,经历了从损伤修复,到非特异性免疫炎症以及特异性免疫炎症研究的三个阶段。从20世纪早期,缺乏对AS复杂生物成分的了解,仅当作动脉管壁内过剩脂质沉积:到70年代,Russell Ross等发现AS是动脉对损伤的慢性炎症修复过程,oxLDL损伤动脉内皮,激活非特异性免疫,大量白细胞、巨噬细胞激活并迁移到动脉内膜,超负载吞噬LDL后破裂坏死,释放出更多毒性脂质。AS作为一种炎症的观点已经得到广泛认可,动脉壁类似于战场,进攻方是oxLDL等毒性物质,防守方是非特异性免疫系统。90年代,Erling Falk、Aarhus和Micheal David等同时提出AS的快速进展取决于斑块的稳定性,并指出不稳定斑块包含更多的脂质、炎症和坏死凋亡的细胞,炎症细胞在病变局部聚集,释放大量炎症因子和活性物质,导致病变局部出现细胞凋亡和基质降解增多,斑块逐渐薄弱不稳定而出现破裂。破裂的粥样斑块可以加速血栓形成,并完全地封闭动脉管腔而阻断血流供应。伴随着不稳定斑块概念的界定,临床上急性冠脉综合症的临床分型也逐渐明确。以不稳定斑块为病变基础,以不稳定心绞痛、非Q波心肌梗死和心肌梗死等临床表现为特征的一类临床症候群,被定义为急性冠脉综合症。除了在病变局部出现炎症细胞大量聚集和细胞因子分泌增多,ACS患者外周血中同样发现中性粒细胞等活化炎症细胞增多和TNF-α、IL-6及CRP等炎症因子的升高。更进一步,K.Hansson等人在斑块中发现激活T淋巴细胞的存在,不久在急性冠脉综合症患者外周血发现处于活化状态的T淋巴细胞增殖。揭开AS在特异性免疫领域的研究高潮。
特异性免疫系统的特征是高度特异性和精确性,相对于非特异性免疫来说,需要更长时间的激活。非特异性免疫细胞攻击所有非己结构,而单克隆T淋巴细胞仅仅识别一个特异性的抗原,并激活特异性的免疫反应。体内有亿万不同克隆的T细胞在血液中流动和淋巴器官内居住。特异性抗原由抗原提呈细胞,例如巨噬细胞、树突状细胞等,提取呈递给特异性的T细胞克隆。当受到特定的抗原刺激后,T细胞克隆开始激活并分化,成为诱导变态反应及抗体合成的Th1细胞和抑制免疫反应及免疫耐受的辅助T细胞。斑块内激活的T细胞以及外周血发现激活的T淋巴细胞,使免疫机制是否参与冠心病的发生发展成为关注的焦点。冠心病究竟与免疫系统激活之间存在什么样的关系,是“父子关系”,还是“兄弟关系”,或者是同行的陌生人呢?AS究竟是特异性免疫还是非特异性的损伤反应,特异性的T细胞克隆活化成为解决问题的核心环节。
外周成熟的T淋巴细胞无论在表型还是功能上都是异质性群体。按照CD分子不同分成CD4+和CD8+两大亚群;按照功能不同,分为辅助性T细胞(help T cell,Th)、细胞毒性T细胞(cytotoxic T cell,Tc)和抑制性T细胞(suppressor T cell,TS)。CD4+Th细胞根据所分泌的细胞因子不同,将其分为Th0,Th1,Th2和Th3三种亚组。抗原刺激后短期内,原始T细胞,即Th0细胞,可以产生多种细胞因子。随后受细胞因子、抗原特性和其他激素等因素的影响,Th0细胞逐渐向Th1和Th2分化。Th1细胞偏向分泌IL-2、IFN-γ和TNF-α,与Tc细胞和TDTH的增殖、分化和成熟有关,主要促进细胞介导的免疫应答;Th2细胞偏向于分泌IL-4、IL-10,与B细胞增殖、成熟和促进抗体的生成有关,故可增强抗体介导的体液免疫。正常情况下,Th1/Th2细胞存在着网络调节,细胞功能趋于动态平衡。病理条件下,Th1/Th2细胞亚组可能出现功能失衡。ACS患者外周血是否存在特异性的T淋巴细胞增殖。首先应该检测其外周血中是否存在T淋巴细胞亚组失平衡。
T细胞受体(T cell receptor,TCR)是T细胞表面的抗原受体。参与抗原的特异性识别。免疫反应的激发均需要CD4+T淋巴细胞首先识别抗原呈递细胞表面的MHC抗原肽复合体,而其识别依赖于抗原特异的、MHC-Ⅱ类限制的TCR,所以TCR一直是T淋巴细胞活化的研究热点。TCR是由α/β或γ/δ组成的异质二聚体结构,两条链之间通过二硫键连接,形成跨膜多肽。在人外周血中大部分的T淋巴细胞(95%左右)表达α/βTCR,因此。α/βTCR是成熟T淋巴细胞的主要抗原识别受体,它通过与特异性抗原的非共价结合,使TCR识别MHC—抗原复合物后的刺激信号传入细胞内。TCR有三个互补决定区(complementarydetermining region,CDR)。其中CDR3是TCR直接与抗原决定簇结合的位点,具有多样性,以适应外界多种多样的抗原。一种CDR3序列代表一个T细胞克隆。研究TCRβ链CDR3谱型能很好地反映不同疾病状态下T细胞的功能,发现疾病相关T细胞克隆。因此,研究是否存在特异性增殖的T淋巴细胞克隆,且是否参与AS的发生发展。我们进一步检测ACS患者外周血T淋巴细胞TCRβ链CDR3谱型。
虽然,迄今为止冠心病的发病机制仍然未能明确,人们在预防和治疗冠心病的过程中倍感艰辛。然而,如此庞大的发病率及致残和致死率,使得人们近百年来竭尽全力寻找减少和控制疾病继续进展的方法和药物,针对AMI的治疗也经历了三个里程碑的进步,取得了一定的成绩。心脏重症监护室和相关监护制度的确立和静脉溶栓治疗的发展,又使得AMI的治疗从被动保守治疗转入了以恢复濒死心肌血流灌注的主动干预治疗,不仅降低AMI的死亡率,并减少了缺血性心脏病和顽固性心力衰竭的发生;近20年来,最令人瞩目的治疗措施就是冠脉介入治疗和强化降脂治疗。介入治疗消除了斑块的物理影响,改变血管狭窄的状态,恢复缺血心肌的灌注;而强化降脂治疗则不仅仅具有降低血脂的作用,并在临床上逐渐显示出在斑块稳定和控制斑块发展上的强大治疗效果。然而,有关他汀类药物非降脂作用的机制仍不是十分清楚。有研究表明,他汀类药物具有调节免疫作用。既然他汀类药物非降脂作用以斑块稳定和控制斑块进展为主要特点,那么他汀类药物是否通过体内的免疫调节和炎症控制起作用,我们随访应用他汀类药物治疗的ACS患者,观察药物治疗对免疫反应的作用,从另一角度再次证实特异性的免疫反应是否参与AS的发生发展。
本实验分为三个部分,分别入选临床上冠脉造影正常的对照组、具有胸痛症状而造影正常的胸痛综合症患者组、稳定性心绞痛组和急性冠脉综合症组,共81例患者。通过酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)分析不同病人外周血Th1/Th2细胞因子水平,及通过培养的T淋巴细胞上清液Th1/Th2细胞因子的检测,分析Th1/Th2细胞亚组之间平衡情况,通过流式细胞检测技术检测外周血CD4+CD28-T细胞亚组和CD4+CD25+T细胞亚组分布。接着采用逆转录聚合酶链反应(reverse transcription-polymerase chain raction,RT-PCR)和基因扫描等方法,检测各组患者外周血T淋巴细胞TCR链CDR3谱型。最后,随访6个月继续观察应用阿托伐他汀治疗后,各组患者T淋巴细胞亚组和外周血T淋巴细胞TCRβ链CDR3谱型变化。结果分述如下:
1.ACS患者外周血T淋巴细胞亚组出现偏移
1.1各组患者一般临床资料情况比较差异无显著性意义
临床入选病例总共81例,其中AMI组20例、UAP组20例、SAP组20例、CPS组11例、对照组10例。一般临床资料的比较显示:各组的年龄、性别比例相比差异无显著性意义(P＞0.05)。各组中主要的心血管病危险因素,如:吸烟(Smoking)、高血压(Hypertension)、糖尿病(Diabetes mellitus)、总胆固醇(Totalcholesterol)、甘油三脂(Triglyceride)、高密度脂蛋白胆固醇(HDL cholesterol)、低密度脂蛋白胆固醇(LDL cholesterol)等,相互之间的比较差异无显著性意义(P＞0.05):
1.2各组患者血浆Th1/Th2细胞因子水平比较,ACS组患者Th1细胞因子水平上升,Th2细胞因子各组无明显差异
正常对照组、CPS组、SAP组、UAP组和AMI组的IL-4表达量分别为5.31±1.4,5.91±1.02,5.7±1.4,5.47±1.1和6.36±1.38pg/ml以及IL-10表达量分别为32.89±9.3,33.16±9.65,32.3±9.44,34.5±11.15和35.2±10.24p/ml。IL-4和IL-10分别在五组间差异无显著性意义(F_(IL-4)=1.682和F_(IL-10)=0.292,均P＞0.05)。正常对照组、CPS组、SAP组、UAP组和AMI组的IFN-γ表达量分别为5.66±2.17,6.35±1.68,6.56±1.50,15.71±2.68和14.27±3.25pg/ml以及TNF-α表达量分别为34.2±6.98,38.4±7.81,36.6±7.56,56.5±9.58和55.7±10.24pg/ml。IFN-γ和TNF-α分别在五组间差异有显著性意义(F_(IFN-γ)=65.219和F_(TNF-α)=24.941,均P＜0.001)。LSD比较分析表明,UAP组和AMI组比正常对照组、CPS组和SAP组Th1(IFN-γ和TNF-α)细胞因子分泌增加(均P＜0.001):
1.3各组患者外周血分离T淋巴细胞培养上清Th1/Th2细胞因子检测,ACS组患者Th1细胞因子水平上升,Th2细胞因子各组差异无显著性意义
正常对照组、CPS组、SAP组、UAP组和AMI组的IL-4表达量分别为60.9±8.7,63.1±10.1,62.6±9.7,63.1±9.8和64.2±10.0pg/ml以及IL-10表达量分别为30.1±6.15,33.7±6.24,32.5±7.21,32.95±7.92和35.2±8.1pg/ml。IL-4和IL-10分别在五组间差异无显著性意义(F_(IL-4)=0.263和F_(IL-10)=0.641,均P＞0.05)。正常对照组、CPS组、SAP组、UAP组和AMI组的IFN-γ表达量分别为1003.95±230.5,991.8±197.2,965.5±250.9,1756.1±306.5和1834.4±314.5pg/ml以及TNF-α表达量分别为476.3±98.1,486.7±115.2,482.2±125.0,861.7±172.3和858.7±189.4pg/ml。IFN-γ和TNF-α分别在五组间差异有显著性意义(F_(IFN-γ)=45.408和F_(TNF-α)=31.338,均P＜0.001)。LSD比较分析表明,UAP组和AMI组比正常对照组、CPS组和SAP组Th1(IFN-γ和TNF-α)细胞因子分泌增加(均P＜0.001);
1.4各组患者外周血CD4+CD28-和CD4+CD25+T淋巴细胞亚组占CD4+T淋巴细胞总数比例检测,ACS组CD4+CD28-比例增多,而CD4+CD25+T淋巴细胞亚组比例降低
正常对照组、CPS组、SAP组、UAP组和AMI组的CD4+CD28-细胞占CD4+淋巴细胞比例分别为2.68±0.75%,2.79±0.49%,2.68±1.71%,10.7±5.3%和10.9±5.97%。CD4+CD25+细胞占CD4+淋巴细胞比例分别为10.29±3.71%,10.24±3.79%,11.4±4.27%,6.43±2.68%和5.9±2.23%。CD4+CD28-细胞和CD4+CD25+细胞分别在五组间差异有显著性意义(F_(CD4+CD28-)=11.795和F_(CD4+CD25+)=15.912,均P＜0.001)。LSD比较分析表明,UAP组和AMI组比正常对照组、CPS组和SAP组CD4+CD28-细胞比例和CD4+CD25+细胞比例差异有显著性意义(均P＜0.001)。
2.ACS组检测到TCRVβ亚家族寡克隆表达数量明显增多
ACS患者中17例患者有一个或多个寡克隆表达的Vβ亚家族存在,而SAP患者中仅有3例患者表达Vβ亚家族的寡克隆,正常对照和CPS患者外周血Vβ亚家族大多都呈多克隆表达,未见单克隆或寡克隆表达。在所有20例存在单克隆或寡克隆表达的患者中,存在Vβ3、Vβ12、Vβ14、Vβ17和Vβ22亚家族寡克隆的患者超过患者总数的25%,分别为6例、5例、6例、7例和5例,而检测出Vβ6.2、Vβ7、Vβ9、Vβ18和Vβ21亚家族寡克隆表达的低于总患者数目的25%,分别为3例、3例、2例、1例和3例。比较检测出Vβ亚家族单克隆表达的患者数,ACS组与SAP、CPS和正常组差异有显著性意义(F=16.9,P＜0.001),而SAP与CPS和正常组差异无显著性意义(F=1.57,P=0.2104)。通过TCRVβ亚家族检测结果,发现在ACS患者外周血,大部分可以检测到单克隆表达的TCRVβ亚家族,且较SAP、CPS和正常对照组具有显著性意义,说明ACS患者外周血可能存在着某一或几个T淋巴细胞的单克隆增殖,这种单克隆T淋巴细胞增殖可能与AS斑块内抗原物质激活免疫细胞有关,并且与炎症反应的扩增与斑块的不稳定高度相关。
3.随访6个月,阿托伐他汀对ACS患者组具有明显免疫调节作用
3.1服用阿托伐他汀6个月后,血脂治疗效果、临床终点事件及冠脉造影复查结果各组比较:
各组患者服用阿托伐他汀20mg 1/日6月后,AMI、UAP和SAP组患者血浆TC分别为3.89±0.26、3.98±0.35和3.01±0.32mmol/L,分别下降31.4±10.8%、22.8±7.3%和35.3±18.8%;低密度脂蛋白分别为2.25±0.35、2.06±0.26、1.85±0.34mmol/L,分别下降36.2±17.7%、37.9±11.8%、37.6±18.9%,与服药前相比差异有显著性意义(P＜0.05),高密度脂蛋白和甘油三脂没有变化。AMI组患者有以1例出现支架内再狭窄,UAP组和SAP组冠脉造影复查无支架内再狭窄等情况,三组的MACE发生情况差异均无显著性意义(P＞0.05):
3.2服用阿托伐他汀6个月后,血浆Th1/Th2因子ELISA检测结果各组比较:
随访复查血浆Th2细胞因子的结果发现SAP组、UAP组和AMI组IL-4表达量分别为5.15±1.72,5.88±1.07和6.38±1.12 pg/ml,以及IL-10表达量分别为35.4±6.03、32.4±5.29和35.4±6.0 pg/ml。与6月前相比,IL-4和IL-10无显著性变化。SAP组、UAP组和AMI组IFN-γ表达量分别为6.07±1.04、6.75±1.02和6.89±1.74pg/ml,TNF-α表达量分别为38.2±6.79、39.5±7.38和40.5±7.46p/ml。与6月前相比,UAP组和AMI组Th1(IFN-γ和TNF-α)细胞因子分泌差异有显著性意义(均P＜0.001),而SAP组降低无统计学差异;
3.3随访各组PBTC培养上清液Th1和Th2细胞因子的变化情况:
对照组、SAP组、UAP组和AMI组的IFN-γ表达量分别为1042.1±253.1,947.47±142.6,1136.4±211.7和1023.6±186.7pg/ml以及TNF-α表达量分别为459.7±131.6,490.88±127.3,511.74±132.8和519.8±159.5pg/ml。与6月前相比,UAP组和AMI组Th1(IFN-γ和TNF-α)细胞因子分泌差异有显著性意义(均P＜0.001),而SAP组降低无统计学差异。正常对照组、SAP组、UAP组和AMI组的IL-4表达量分别为67.37±5.17,61.8±14.02,66.39±14.14和66.95±16.06pg/ml以及IL-10表达量分别为32.5±6.81,35.3±8.2,36.3±7.94和39.76±8.93pg/ml。与6月前相比,各组患者IL-4和IL-10分别在三组间差异无显著性意义。
3.4各组患者外周血CD4+CD28-和CD4+CD25+T淋巴细胞亚组占CD4+T淋巴细胞总数比例情况
正常对照组、SAP组、UAP组和AMI组的CD4+CD28-细胞占CD4+淋巴细胞比例分别为2.6±0.5%,3.7±0.94%,3.2±1.28%和3.29±0.9%。CD4+CD25+细胞占CD4+淋巴细胞比例分别为10.05±4.95%,10.4±2.12%,10.57±2.12%和10.24±2.03%。与服用阿托伐他汀药物前相比,UAP组和AMI组随访CD4+CD28-细胞数量比例和CD4+CD25+细胞比例差异有显著性意义(均P＜0.001);
3.5阿托伐他汀服用后对TCRVβ亚家族寡克隆表达的作用:
ACS组中原检测有一个或多个寡克隆表达的Vβ亚家族存在的17例患者,随访时仅9例患者仍可以检测到与前检测相似的Vβ亚家族寡克隆的表达,其他患者趋向于多克隆表达;而SAP组原来检测存在Vβ亚家族寡克隆表达的3例患者,有2例仍可以检测到与前相似的寡克隆表达。比较检测出Vβ亚家族单克隆表达的患者数,ACS组与SAP组差异无显著性意义(F=0.88,P=0.3484)。
通过上述三个部分的实验,我们能够得出以下结论:①ACS患者外周血Th1/Th2细胞因子发生改变,ACS患者体内存在免疫系统的激活:②ACS患者外周血PBTC培养上清中Th1细胞因子分泌增多,Th1细胞处于活化状态;③ACS患者外周血中CD4+CD28-和CD4+CD25+T淋巴细胞亚组发生比例变化,T淋巴细胞活化及不均衡诱导,可能与斑块不稳定有关;④ACS患者外周血T淋巴细胞表面TCRVβ亚家族寡克隆表达数量增多,独特抗原表型T淋巴细胞单克隆增殖明显,抗原呈递与斑块的不稳定高度相关;⑤阿托伐他汀可以降低ACS患者外周血Th1细胞因子的浓度;降低ACS患者外周血T淋巴细胞分泌Th1细胞因子;调节ACS患者外周血CD4+CD28-和CD4+CD25+T淋巴细胞比例,使T淋巴细胞亚组趋于平衡;使得ACS患者T淋巴细胞表面TCRVβ亚家族寡克隆表达数量减少并趋于多克隆表达,通过调控抗原呈递达到抗炎和免疫调节作用,用于防治AS的发生发展具有广阔的前景。
Atherosclerosis(AS)is the main pathological process of coronary heart disease(CHD),cerebrovascular accident and peripheral vascular disease.In this century,it is presently one of the most harmful human diseases with higheat morbidity.In order to conquer this disease,many medical groups have paid attention to a series of research on the aspects of pathology,epidemiology and etiology,but the main mechanism of AS is still unknown.
Take a wide view on the study process of AS,it has experienced three stages from vascular repair to nonspecific and specific immunity.In the early of 20 century, AS was regarded as excess lipidoses on the arterial canal.To the 70's,Russell Ross found out the reparative process and chronic inflammation might be the main point. The damage of oxLDL on the endothelium could induce the activity of nonspecific immunity system.And a lot of leucocytes and macrophages were stimulated to migrate through the endarterium,swallowed oxLDL and overloaded to die.The toxic lipid was released to stimulate more cells.It has been generally proved that AS is the process of chronic inflammation.The artery is similar to the battlefield,toxic substances,such as oxLDL,act as attacker,and the defense is nonspecific immunity system.In the 90's,Erling Falk,Aarhus and Micheal David almost proposed that the rapid progress of AS was highly correlative with the unstability of plaque in the same time,they also indicated that unstable plaque may be comprised of more lipoids,inflammation and dead cells.Inflammatory cells gathered in the plaque,and released a great quantity of inflammatory factors and active substances.This would lead to cell necrosis and matrix degradation,and then the plaque became more unstable.Thrombosis would form and block up the blood supply,if the plaque was ruptured.And this was regarded as acute coronary syndrome,a clinic syndrome defined with the characteristic of unstable plaque and clinical manifestation of unstable angina and myocardial infarction.It has been found that neutrophilic leukocytes in the peripheral blood are increased and the serum level of TNF-α,IL-6 and CRP are also elevated in the ACS patients.Furthermore,K.Hansson found that activated T cells existed in the plaque,even in the peripheral blood were also detected.More interest was paid on the relationship between the specific immunity system and AS.
The specific immunity is more specific and exact than the nonspecific immunity. The nonspecific immunity assaults with all of the material out of our own,but monoclonal T cells just recognize one specific antigen.Hundreds of millions kinds of T cells are flowing with blood and dwelling in the lymphatic organ.Specific antigen could be recognize and extracted by antigen presenting cells(APC),such as macrophagocytes and dendritic cells et al.When specific T cell clone accepts the signal from antigen presenting cells,it will be activated and differentiate.The specific T cell clone could differentiate into Th cell which can induce immune responses and Ts cell which lead to immunodepressive.The exist of activated T cells in the plaque and peripheral blood suggests that immunologic mechanism may play the important role in the genesis and development of AS.The relationship between the immune system and AS is father-child relation or brother relation.And the exist of activated specific T cells clone become the core element of the question whether AS finally belong to specific immune or nonspecific immune.
T cells in the peripheral blood could be divided into different kinds of subgroups according to their phenotype and function.Usually two subgroups of CD4+ T cell and CD8+ T cell is firstly be separated on their phenotype of CD expression.And T cells can also be divided into three subgroups of help T cell(Th), cytotoxic T cell(Tc)and suppressor T cell(Ts)on their different functions.CD4+ Th cells can be classified into three subgroups of Th0,Th1 and Th2 according to the cytokine secreted.The native T cells,I.e.Th0 cell,can produce many kinds of cytokine soon after stimulated by antigen.And then Th0 cell will differentiate to Th1 and Th2 cell according to the different influence of cytokines,antigen characteristic and other hormones.Th1 cells could synthesize some cytokines such as IL-2,IFN-γand TNF-α,and promote the cellular immune.Although Th2 cells could enhance the antibody mediated humoral immunity by the secretion of cytokines of IL-4 and IL-10.In the normal circumstance,Th1/Th2 cells keep the voluntary balance to maintain proper functions.But in the pathological conditions,the balance may be broken down,the disequilibrium of subgroups possibly lead to immune disorder.In order to illuminate specific antigen activated T cells clone expands in AS,we should firstly detect whether the unbalance of T cells subgroups exist or not in the peripheral blood.
T cell receptor(TCR)is the receptor of the specific antigen on the surface of T cell.The main function of TCR is antigen recognition.The beginning of immune response was based on the TCR on the CD4+ T cell to recognize the MHC-antigen compounds on the APC surface.TCR has always been proven to be the core point of the research on the stimulation of T cells.TCR is a dimeride structure consisted ofα/βorγ/δsubunit.Two peptide chains connected with disulfide bond form transmembrance polypeptides.In our peripheral bloodα/βpeptide chains is expressed by most of T cells(95%),soα/βTCR is the main antigen recognized receptor on the mature T cells,α/βTCR can distinguish the specific antigen-MHC compounds,combine with noncovalent interaction,and the stimulated signal conducts into cellular in the last.TCR consists with three complementary determining region(CDR).Among them,CDR3 is just the binding site with the antigen determinant.And CDR3 is the hypervariable region of TCR adapted with all kinds of antigen outside.That to say,different kinds of CDR3 sequences maybe represent different T cell clone.The study on the sequence of CDR3 has already proven to be the well tools reflecting the function of T cells.In order to find the expands of specific T cell clone corresponded with unknown disease,we should detect the sequence of CDR3 in the PBTC from ACS patients.
Although until now the mechanism of AS is still unknown,it is too difficult in the fight with CHD.Scientists have tried their best to find the methods and drug to prevent and control the process of AS for the past century.Three main progresses were experienced in the treatment of acute myocardial infarction.The establishment of institution of cardiac intensive care and the treatment of intravenous thrombolytic therapy change the CHD treatment from conservative therapy to active therapy to pursue the best result for more survival myocardial muscle.It has been believed that the active therapy not only turns down the mortality of AMI,but also decreases the incidence of ischemia heart disease and refractory heart failure.In the past 20 years,the main noticeable improvements are coronary intervention treatment and intensive lipid lowing therapy.The intervention treatment eliminates the blockage of plaque,change the narrow situation and recover the blood perfusion. And intensive lipid lowing therapy could stabilize the plaque and control the process of AS unless lowing the hyperlipemia.But the mechanism of pleiotropic effect of statins is still unknown.Some study found that statins may have the potential capability of immunological regulation.The inflammatory reaction is the main cause of unstable plaque,and then statins could stabilize the plaque.We presume that statins maybe control the process the unstable plaque by the function of immunoregulation.So we present a follow up research on the ACS patients after six month lowing lipid therapy to observe the effect on the immune system of statins.It is expected to prove the relationships between the specific immune and the process of AS.
In this study,we recruited 81 hospital patients from Jan 2006 to July 2006 in zhujiang hospital.All the patients were divided into five group:control group(Control),chest pain syndrome group with normal coronary angiograph(CPS), stable angina group(SAP),unstable angina group(UAP)and acute myocardial infarction group(AMI).In the first part,the level of Th1/Th2 cytokine in the peripheral serum and the culture supematant of PBTC were detected by enzyme-linked immunosorbent assay(ELISA).The ratio of CD4+CD28- and CD4+CD25- T cell subgroups were checked by flow cytometry(FCM).The main purpose was to analyze the change of the balance of Th1/Th2 subgroups and other subgroups and to find out the clue that activated T cells exist in the peripheral blood of AS patients.In the second part,the sequences of TCR Vβchain were analyzed by reverse transcription polymerase chain raction(RT-PCR)and gene scan to confirm the expand of monoclonal T cells.At last,we performed a follow up of statins treatment of six months to observe the change of items above,and affirmed the immuno- regulation of statins.We got the result as follows:
1.The balance of T cell subgroups were broken in the peripheral blood of ACS patients
1.1 Baseline clinical characteristics in patients with CHD and healthy control subjects
All the 81 patients recruited were separated into AMI group(n=20),UAP group(n=20),SAP group(n=20),CPS group(n=11)and Control group(n=10).There is no significant difference in the baseline clinical characteristics in all of the groups(P＜0.05),such as:age;gender;smoking;the ratio of hypertension and diabetes mellitus;the serum level of total cholesterol(TC),triglyceride(TG),HDL and LDL,et al.
1.2 The serum level of Th1/Th2 cytokines in all of groups
The serum level of IL-4 in all groups was detected as 5.31±1.4,5.91±1.02, 5.7±1.4,5.47±1.1 and 6.36±1.38pg/ml,IL-10 was 32.894±9.3,33.16±9.65,32.34±9.44, 34.5±11.15 and 35.24±10.24 pg/ml.There is no significant difference among all of the groups(P＞0.05).The serum level of IFN-γin control,CPS,SAP,UAP and AMI groups was detected as 5.664±2.17,6.354±1.68,6.564±1.50,15.714±2.68 and 14.274±3.25 pg/ml and TNF-αwas 34.24±6.98,38.4±7.81,36.64±7.56,56.54±9.58 and 55.7±10.24 pg/ml.The level of IFN-γand TNF-αwere significant elevated in UAP and AMI groups than other groups(P＜0.001).
1.3 The level of Th1/Th2 cytokines in the culture supematants of PBTC of all groups
The level of IL-4 in culture supernatants of PBTC was detected as 60.94±8.7, 63.1±10.1,62.64±9.7,63.14±9.8 and 64.2±10.0 pg/ml,IL-10 was 30.14±6.15,33.7±6.24, 32.54±7.21,32.954±7.92 and 35.2±8.1pg/ml.There is no significant difference among all of the groups(P＞0.05).The level of IFN-γin culture supernatants of PBTC in control,CPS,SAP,UAP and AMI groups was detected as 1003.954±230.5, 991.84±197.2,965.5±250.9,1756.14±306.5 and 1834.44±314.5 pg/ml and TNF-αwas 476.34±98.1,486.74±115.2,482.24±125.0,861.74±172.3 and 858.74±189.4 pg/ml.The level of IFN-γand TNF-αwere significant elevated in the culture supematants of PBTC of UAP and AMI groups than other groups(P＜0.001).
1.4 The ratio of CD4+CD28- and CD4+CD25- T cell subgroups in all of the groups
The ratio of CD4+CD28- / CD4+ T cells in control,CPS,SAP,UAP and AMI groups was 2.68±0.75%,2.79±0.49%,2.68±1.71%,10.74±5.3%and 10.94±5.97%; the ratio of CD4+CD25+/CD4+ was respectively 10.29±3.71%,10.2±3.79%, 11.4±4.27%,6.43±2.68%and 5.9±2.23%.The ratio of CD4+CD28- T cell subgroups was significant increased in the peripheral blood of ACS groups;and the ratio of CD4+CD25+ T cell subgroups was decreased in ACS groups.
2.The expression of monoclonal TCR Vβsubfamilies in ACS groups
There are more than one monoclonal TCR Vβsubfamilies which can be detected in 17 cases of ACS groups;but in only three patients of SAP groups.All of the TCR Vβsubfamilies were expressed in the polyclonal way in the patients of Normal and CPS groups.In all the 20 cases of monoclonal expression,the number of patients with the expression of Vβ3,Vβ12,Vβ14,Vβ17 and Vβ22 subfamilies were more than 25%of all the cases,and were respectively 6cases,5cases,6cases, 7cases and 5cases;the number of patients with expression of Vβ6.2,Vβ7,Vβ9,Vβ18 and Vβ21 subfamilies were less than 25%,and were repectively 3cases, 3cases,2cases,1eases and 3cases.The numbers of patients with monoclonal subfamilies in ACS group were more than in other groups significantly.
3 The immunoregulation function of atorvastatin on the six month follow up
3.1 Effect of therapy of atorvastatin on patients with CAD after six month follow up
After six months follow up,the serum level of TC in AMI,UAP and SAP groups was 3.89±0.26,3.98±0.35 and 3.01±0.32mmol/L,was decreased in 31.4±10.8%,22.8±7.3%and 35.3±18.8%,respectively.The serum level of LDL was 2.25±0.35,2.064±0.26 and 1.85±0.34mmol/L,and was decrease in 36.2±17.7%,37.9±11.8%and 37.6±18.9%.The level of TG and HDL were not changed remarkable. Only 1 patient in AMI groups was found with instent restenosis.There are no discrepancy in incidence rate of MACE in all groups followed up.
3.2 Effect of atorvastatin on the serum level of Th1/Th2 cytokines in all of groups
The serum level of IL-4 in SAP,UAP and AMI groups was detected as 5.15±1.72,5.88±1.07 and 6.38±1.12pg/ml,IL-10 was为35.44±6.03,32.4±5.29 and 35.4±6.0 pg/ml.There is no significant difference compared with six months before (P＞0.05).The serum level of IFN-γin SAP,UAP and AMI groups was detected as为6.07±1.04,6.75±1.02 and 6.89±1.74 pg/ml and TNF-αwas38.2±6.79,39.5±7.38 and 40.5±7.46 pg/ml.The level of IFN-γand TNF-αwere significant decrease in UAP and AMI groups compared with six months before(P＜0.001).
3.3 Effect of atorvastatin on the level of Th1/Th2 cytokines in the culture supematants of PBTC of all groups
The level of IL-4 in culture supernatants of PBTC in control,SAP,UAP and AMI groups was detected as 67.37±5.17,61.8±14.02,66.39±14.14 and 66.95±16.06 pg/ml,IL-10 was 32.5±6.81,35.3±8.2,36.3±7.94 and 39.76±8.93pg/ml.There is no significant difference among all of the groups compared with six month before (P＞0.05).The level of IFN-γin culture supematants of PBTC in control,SAP,UAP and AMI groups was detected as 1042.1±253.1,947.47±142.6,1136.4±211.7 and 1023.6±186.7pg/ml and TNF-αwas 459.7±131.6,490.88±127.3,511.74±132.8 and 519.8±159.5pg/ml.The level of IFN-γand TNF-αwere significant decreased in the culture supematants of PBTC of UAP and AMI groups compared with six months before(P＜0.001).
3.4 Effect of storvastatin on the ratio of CD4+CD28- and CD4+CD25- T cell subgroups in all of the groups
The ratio of CD4+CD28- / CD4+ T cells in control,SAP,UAP and AMI groups was 2.6±0.5%,3.7±0.94%,3.2±1.28%and 3.29±0.9%;the ratio of CD4+CD25+/CD4+ was respectively 10.05±4.95%,10.4±2.12%,10.57±2.12%and 10.24±2.03%.The ratio of CD4+CD28- T cell subgroups of ACS groups was significant decreased in the peripheral blood and the ratio of CD4+CD25+ T cell subgroups was increased compared with six months before.
3.5 Effect of storvastatin on The expression of monoclonal TCR Vβsubfamilies in ACS groups
Only 9 cases in all 17 patients of ACS groups with monoclonal expressions before were detected the same subfamilies expression after six months follow up,the remain 8 cases were in the polyclonal expression's way.In SAP group,2 cases of all the three patients with monoclonal expressions before also can detected the same subfamilies expression.The number of patients of monoclonal expression in the ACS and SAP group were comparable(P=0.3484).
Through experiments of the above three parts,we can make the following conclusions:
①The serum level of Th1/Th2 cytokines was changed in ACS patients,immune response may be conrespond with the pathological machenism of AS;
②The level of T Th1/Th2 cytokines in the culture supematants of PBTC in ACS patients was in the unbalance way,Th1 cell was in the active condition in ACS patients;
③The ratio of CD4+CD28- and CD4+CD25+ T cell subgroups were changed in ACS patients,the active T cell and the unbalance in the proliferation of T cell subgroups may play the important role in the formation of unstable plaque;
④The expression of the monoclonal subfamilies of TCR Vβwere increased in ACS patients,the proliferation of monoclonal T cell increase revealed that antigen, presentation was more relatively with the unstable plaque.
⑤Statins can intervene the proliferation of monoclonal T cells,the secretion of Th1 CKs,and the subgroups balance.These effects provide a theorical foundation for using the statin to act as an immune inhibitor,and have the effects of regulating immunity and anti-inflammation.Thus,the foreground of using statins to prevent and cure AS is quite vast.
纵观AS的研究过程,经历了从损伤修复,到非特异性免疫炎症以及特异性免疫炎症研究的三个阶段。从20世纪早期,缺乏对AS复杂生物成分的了解,仅当作动脉管壁内过剩脂质沉积:到70年代,Russell Ross等发现AS是动脉对损伤的慢性炎症修复过程,oxLDL损伤动脉内皮,激活非特异性免疫,大量白细胞、巨噬细胞激活并迁移到动脉内膜,超负载吞噬LDL后破裂坏死,释放出更多毒性脂质。AS作为一种炎症的观点已经得到广泛认可,动脉壁类似于战场,进攻方是oxLDL等毒性物质,防守方是非特异性免疫系统。90年代,Erling Falk、Aarhus和Micheal David等同时提出AS的快速进展取决于斑块的稳定性,并指出不稳定斑块包含更多的脂质、炎症和坏死凋亡的细胞,炎症细胞在病变局部聚集,释放大量炎症因子和活性物质,导致病变局部出现细胞凋亡和基质降解增多,斑块逐渐薄弱不稳定而出现破裂。破裂的粥样斑块可以加速血栓形成,并完全地封闭动脉管腔而阻断血流供应。伴随着不稳定斑块概念的界定,临床上急性冠脉综合症的临床分型也逐渐明确。以不稳定斑块为病变基础,以不稳定心绞痛、非Q波心肌梗死和心肌梗死等临床表现为特征的一类临床症候群,被定义为急性冠脉综合症。除了在病变局部出现炎症细胞大量聚集和细胞因子分泌增多,ACS患者外周血中同样发现中性粒细胞等活化炎症细胞增多和TNF-α、IL-6及CRP等炎症因子的升高。更进一步,K.Hansson等人在斑块中发现激活T淋巴细胞的存在,不久在急性冠脉综合症患者外周血发现处于活化状态的T淋巴细胞增殖。揭开AS在特异性免疫领域的研究高潮。
特异性免疫系统的特征是高度特异性和精确性,相对于非特异性免疫来说,需要更长时间的激活。非特异性免疫细胞攻击所有非己结构,而单克隆T淋巴细胞仅仅识别一个特异性的抗原,并激活特异性的免疫反应。体内有亿万不同克隆的T细胞在血液中流动和淋巴器官内居住。特异性抗原由抗原提呈细胞,例如巨噬细胞、树突状细胞等,提取呈递给特异性的T细胞克隆。当受到特定的抗原刺激后,T细胞克隆开始激活并分化,成为诱导变态反应及抗体合成的Th1细胞和抑制免疫反应及免疫耐受的辅助T细胞。斑块内激活的T细胞以及外周血发现激活的T淋巴细胞,使免疫机制是否参与冠心病的发生发展成为关注的焦点。冠心病究竟与免疫系统激活之间存在什么样的关系,是“父子关系”,还是“兄弟关系”,或者是同行的陌生人呢?AS究竟是特异性免疫还是非特异性的损伤反应,特异性的T细胞克隆活化成为解决问题的核心环节。
外周成熟的T淋巴细胞无论在表型还是功能上都是异质性群体。按照CD分子不同分成CD4+和CD8+两大亚群;按照功能不同,分为辅助性T细胞(help T cell,Th)、细胞毒性T细胞(cytotoxic T cell,Tc)和抑制性T细胞(suppressor T cell,TS)。CD4+Th细胞根据所分泌的细胞因子不同,将其分为Th0,Th1,Th2和Th3三种亚组。抗原刺激后短期内,原始T细胞,即Th0细胞,可以产生多种细胞因子。随后受细胞因子、抗原特性和其他激素等因素的影响,Th0细胞逐渐向Th1和Th2分化。Th1细胞偏向分泌IL-2、IFN-γ和TNF-α,与Tc细胞和TDTH的增殖、分化和成熟有关,主要促进细胞介导的免疫应答;Th2细胞偏向于分泌IL-4、IL-10,与B细胞增殖、成熟和促进抗体的生成有关,故可增强抗体介导的体液免疫。正常情况下,Th1/Th2细胞存在着网络调节,细胞功能趋于动态平衡。病理条件下,Th1/Th2细胞亚组可能出现功能失衡。ACS患者外周血是否存在特异性的T淋巴细胞增殖。首先应该检测其外周血中是否存在T淋巴细胞亚组失平衡。
T细胞受体(T cell receptor,TCR)是T细胞表面的抗原受体。参与抗原的特异性识别。免疫反应的激发均需要CD4+T淋巴细胞首先识别抗原呈递细胞表面的MHC抗原肽复合体,而其识别依赖于抗原特异的、MHC-Ⅱ类限制的TCR,所以TCR一直是T淋巴细胞活化的研究热点。TCR是由α/β或γ/δ组成的异质二聚体结构,两条链之间通过二硫键连接,形成跨膜多肽。在人外周血中大部分的T淋巴细胞(95%左右)表达α/βTCR,因此。α/βTCR是成熟T淋巴细胞的主要抗原识别受体,它通过与特异性抗原的非共价结合,使TCR识别MHC—抗原复合物后的刺激信号传入细胞内。TCR有三个互补决定区(complementarydetermining region,CDR)。其中CDR3是TCR直接与抗原决定簇结合的位点,具有多样性,以适应外界多种多样的抗原。一种CDR3序列代表一个T细胞克隆。研究TCRβ链CDR3谱型能很好地反映不同疾病状态下T细胞的功能,发现疾病相关T细胞克隆。因此,研究是否存在特异性增殖的T淋巴细胞克隆,且是否参与AS的发生发展。我们进一步检测ACS患者外周血T淋巴细胞TCRβ链CDR3谱型。
虽然,迄今为止冠心病的发病机制仍然未能明确,人们在预防和治疗冠心病的过程中倍感艰辛。然而,如此庞大的发病率及致残和致死率,使得人们近百年来竭尽全力寻找减少和控制疾病继续进展的方法和药物,针对AMI的治疗也经历了三个里程碑的进步,取得了一定的成绩。心脏重症监护室和相关监护制度的确立和静脉溶栓治疗的发展,又使得AMI的治疗从被动保守治疗转入了以恢复濒死心肌血流灌注的主动干预治疗,不仅降低AMI的死亡率,并减少了缺血性心脏病和顽固性心力衰竭的发生;近20年来,最令人瞩目的治疗措施就是冠脉介入治疗和强化降脂治疗。介入治疗消除了斑块的物理影响,改变血管狭窄的状态,恢复缺血心肌的灌注;而强化降脂治疗则不仅仅具有降低血脂的作用,并在临床上逐渐显示出在斑块稳定和控制斑块发展上的强大治疗效果。然而,有关他汀类药物非降脂作用的机制仍不是十分清楚。有研究表明,他汀类药物具有调节免疫作用。既然他汀类药物非降脂作用以斑块稳定和控制斑块进展为主要特点,那么他汀类药物是否通过体内的免疫调节和炎症控制起作用,我们随访应用他汀类药物治疗的ACS患者,观察药物治疗对免疫反应的作用,从另一角度再次证实特异性的免疫反应是否参与AS的发生发展。
本实验分为三个部分,分别入选临床上冠脉造影正常的对照组、具有胸痛症状而造影正常的胸痛综合症患者组、稳定性心绞痛组和急性冠脉综合症组,共81例患者。通过酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)分析不同病人外周血Th1/Th2细胞因子水平,及通过培养的T淋巴细胞上清液Th1/Th2细胞因子的检测,分析Th1/Th2细胞亚组之间平衡情况,通过流式细胞检测技术检测外周血CD4+CD28-T细胞亚组和CD4+CD25+T细胞亚组分布。接着采用逆转录聚合酶链反应(reverse transcription-polymerase chain raction,RT-PCR)和基因扫描等方法,检测各组患者外周血T淋巴细胞TCR链CDR3谱型。最后,随访6个月继续观察应用阿托伐他汀治疗后,各组患者T淋巴细胞亚组和外周血T淋巴细胞TCRβ链CDR3谱型变化。结果分述如下:
1.ACS患者外周血T淋巴细胞亚组出现偏移
1.1各组患者一般临床资料情况比较差异无显著性意义
临床入选病例总共81例,其中AMI组20例、UAP组20例、SAP组20例、CPS组11例、对照组10例。一般临床资料的比较显示:各组的年龄、性别比例相比差异无显著性意义(P＞0.05)。各组中主要的心血管病危险因素,如:吸烟(Smoking)、高血压(Hypertension)、糖尿病(Diabetes mellitus)、总胆固醇(Totalcholesterol)、甘油三脂(Triglyceride)、高密度脂蛋白胆固醇(HDL cholesterol)、低密度脂蛋白胆固醇(LDL cholesterol)等,相互之间的比较差异无显著性意义(P＞0.05):
1.2各组患者血浆Th1/Th2细胞因子水平比较,ACS组患者Th1细胞因子水平上升,Th2细胞因子各组无明显差异
正常对照组、CPS组、SAP组、UAP组和AMI组的IL-4表达量分别为5.31±1.4,5.91±1.02,5.7±1.4,5.47±1.1和6.36±1.38pg/ml以及IL-10表达量分别为32.89±9.3,33.16±9.65,32.3±9.44,34.5±11.15和35.2±10.24p/ml。IL-4和IL-10分别在五组间差异无显著性意义(F_(IL-4)=1.682和F_(IL-10)=0.292,均P＞0.05)。正常对照组、CPS组、SAP组、UAP组和AMI组的IFN-γ表达量分别为5.66±2.17,6.35±1.68,6.56±1.50,15.71±2.68和14.27±3.25pg/ml以及TNF-α表达量分别为34.2±6.98,38.4±7.81,36.6±7.56,56.5±9.58和55.7±10.24pg/ml。IFN-γ和TNF-α分别在五组间差异有显著性意义(F_(IFN-γ)=65.219和F_(TNF-α)=24.941,均P＜0.001)。LSD比较分析表明,UAP组和AMI组比正常对照组、CPS组和SAP组Th1(IFN-γ和TNF-α)细胞因子分泌增加(均P＜0.001):
1.3各组患者外周血分离T淋巴细胞培养上清Th1/Th2细胞因子检测,ACS组患者Th1细胞因子水平上升,Th2细胞因子各组差异无显著性意义
正常对照组、CPS组、SAP组、UAP组和AMI组的IL-4表达量分别为60.9±8.7,63.1±10.1,62.6±9.7,63.1±9.8和64.2±10.0pg/ml以及IL-10表达量分别为30.1±6.15,33.7±6.24,32.5±7.21,32.95±7.92和35.2±8.1pg/ml。IL-4和IL-10分别在五组间差异无显著性意义(F_(IL-4)=0.263和F_(IL-10)=0.641,均P＞0.05)。正常对照组、CPS组、SAP组、UAP组和AMI组的IFN-γ表达量分别为1003.95±230.5,991.8±197.2,965.5±250.9,1756.1±306.5和1834.4±314.5pg/ml以及TNF-α表达量分别为476.3±98.1,486.7±115.2,482.2±125.0,861.7±172.3和858.7±189.4pg/ml。IFN-γ和TNF-α分别在五组间差异有显著性意义(F_(IFN-γ)=45.408和F_(TNF-α)=31.338,均P＜0.001)。LSD比较分析表明,UAP组和AMI组比正常对照组、CPS组和SAP组Th1(IFN-γ和TNF-α)细胞因子分泌增加(均P＜0.001);
1.4各组患者外周血CD4+CD28-和CD4+CD25+T淋巴细胞亚组占CD4+T淋巴细胞总数比例检测,ACS组CD4+CD28-比例增多,而CD4+CD25+T淋巴细胞亚组比例降低
正常对照组、CPS组、SAP组、UAP组和AMI组的CD4+CD28-细胞占CD4+淋巴细胞比例分别为2.68±0.75%,2.79±0.49%,2.68±1.71%,10.7±5.3%和10.9±5.97%。CD4+CD25+细胞占CD4+淋巴细胞比例分别为10.29±3.71%,10.24±3.79%,11.4±4.27%,6.43±2.68%和5.9±2.23%。CD4+CD28-细胞和CD4+CD25+细胞分别在五组间差异有显著性意义(F_(CD4+CD28-)=11.795和F_(CD4+CD25+)=15.912,均P＜0.001)。LSD比较分析表明,UAP组和AMI组比正常对照组、CPS组和SAP组CD4+CD28-细胞比例和CD4+CD25+细胞比例差异有显著性意义(均P＜0.001)。
2.ACS组检测到TCRVβ亚家族寡克隆表达数量明显增多
ACS患者中17例患者有一个或多个寡克隆表达的Vβ亚家族存在,而SAP患者中仅有3例患者表达Vβ亚家族的寡克隆,正常对照和CPS患者外周血Vβ亚家族大多都呈多克隆表达,未见单克隆或寡克隆表达。在所有20例存在单克隆或寡克隆表达的患者中,存在Vβ3、Vβ12、Vβ14、Vβ17和Vβ22亚家族寡克隆的患者超过患者总数的25%,分别为6例、5例、6例、7例和5例,而检测出Vβ6.2、Vβ7、Vβ9、Vβ18和Vβ21亚家族寡克隆表达的低于总患者数目的25%,分别为3例、3例、2例、1例和3例。比较检测出Vβ亚家族单克隆表达的患者数,ACS组与SAP、CPS和正常组差异有显著性意义(F=16.9,P＜0.001),而SAP与CPS和正常组差异无显著性意义(F=1.57,P=0.2104)。通过TCRVβ亚家族检测结果,发现在ACS患者外周血,大部分可以检测到单克隆表达的TCRVβ亚家族,且较SAP、CPS和正常对照组具有显著性意义,说明ACS患者外周血可能存在着某一或几个T淋巴细胞的单克隆增殖,这种单克隆T淋巴细胞增殖可能与AS斑块内抗原物质激活免疫细胞有关,并且与炎症反应的扩增与斑块的不稳定高度相关。
3.随访6个月,阿托伐他汀对ACS患者组具有明显免疫调节作用
3.1服用阿托伐他汀6个月后,血脂治疗效果、临床终点事件及冠脉造影复查结果各组比较:
各组患者服用阿托伐他汀20mg 1/日6月后,AMI、UAP和SAP组患者血浆TC分别为3.89±0.26、3.98±0.35和3.01±0.32mmol/L,分别下降31.4±10.8%、22.8±7.3%和35.3±18.8%;低密度脂蛋白分别为2.25±0.35、2.06±0.26、1.85±0.34mmol/L,分别下降36.2±17.7%、37.9±11.8%、37.6±18.9%,与服药前相比差异有显著性意义(P＜0.05),高密度脂蛋白和甘油三脂没有变化。AMI组患者有以1例出现支架内再狭窄,UAP组和SAP组冠脉造影复查无支架内再狭窄等情况,三组的MACE发生情况差异均无显著性意义(P＞0.05):
3.2服用阿托伐他汀6个月后,血浆Th1/Th2因子ELISA检测结果各组比较:
随访复查血浆Th2细胞因子的结果发现SAP组、UAP组和AMI组IL-4表达量分别为5.15±1.72,5.88±1.07和6.38±1.12 pg/ml,以及IL-10表达量分别为35.4±6.03、32.4±5.29和35.4±6.0 pg/ml。与6月前相比,IL-4和IL-10无显著性变化。SAP组、UAP组和AMI组IFN-γ表达量分别为6.07±1.04、6.75±1.02和6.89±1.74pg/ml,TNF-α表达量分别为38.2±6.79、39.5±7.38和40.5±7.46p/ml。与6月前相比,UAP组和AMI组Th1(IFN-γ和TNF-α)细胞因子分泌差异有显著性意义(均P＜0.001),而SAP组降低无统计学差异;
3.3随访各组PBTC培养上清液Th1和Th2细胞因子的变化情况:
对照组、SAP组、UAP组和AMI组的IFN-γ表达量分别为1042.1±253.1,947.47±142.6,1136.4±211.7和1023.6±186.7pg/ml以及TNF-α表达量分别为459.7±131.6,490.88±127.3,511.74±132.8和519.8±159.5pg/ml。与6月前相比,UAP组和AMI组Th1(IFN-γ和TNF-α)细胞因子分泌差异有显著性意义(均P＜0.001),而SAP组降低无统计学差异。正常对照组、SAP组、UAP组和AMI组的IL-4表达量分别为67.37±5.17,61.8±14.02,66.39±14.14和66.95±16.06pg/ml以及IL-10表达量分别为32.5±6.81,35.3±8.2,36.3±7.94和39.76±8.93pg/ml。与6月前相比,各组患者IL-4和IL-10分别在三组间差异无显著性意义。
3.4各组患者外周血CD4+CD28-和CD4+CD25+T淋巴细胞亚组占CD4+T淋巴细胞总数比例情况
正常对照组、SAP组、UAP组和AMI组的CD4+CD28-细胞占CD4+淋巴细胞比例分别为2.6±0.5%,3.7±0.94%,3.2±1.28%和3.29±0.9%。CD4+CD25+细胞占CD4+淋巴细胞比例分别为10.05±4.95%,10.4±2.12%,10.57±2.12%和10.24±2.03%。与服用阿托伐他汀药物前相比,UAP组和AMI组随访CD4+CD28-细胞数量比例和CD4+CD25+细胞比例差异有显著性意义(均P＜0.001);
3.5阿托伐他汀服用后对TCRVβ亚家族寡克隆表达的作用:
ACS组中原检测有一个或多个寡克隆表达的Vβ亚家族存在的17例患者,随访时仅9例患者仍可以检测到与前检测相似的Vβ亚家族寡克隆的表达,其他患者趋向于多克隆表达;而SAP组原来检测存在Vβ亚家族寡克隆表达的3例患者,有2例仍可以检测到与前相似的寡克隆表达。比较检测出Vβ亚家族单克隆表达的患者数,ACS组与SAP组差异无显著性意义(F=0.88,P=0.3484)。
通过上述三个部分的实验,我们能够得出以下结论:①ACS患者外周血Th1/Th2细胞因子发生改变,ACS患者体内存在免疫系统的激活:②ACS患者外周血PBTC培养上清中Th1细胞因子分泌增多,Th1细胞处于活化状态;③ACS患者外周血中CD4+CD28-和CD4+CD25+T淋巴细胞亚组发生比例变化,T淋巴细胞活化及不均衡诱导,可能与斑块不稳定有关;④ACS患者外周血T淋巴细胞表面TCRVβ亚家族寡克隆表达数量增多,独特抗原表型T淋巴细胞单克隆增殖明显,抗原呈递与斑块的不稳定高度相关;⑤阿托伐他汀可以降低ACS患者外周血Th1细胞因子的浓度;降低ACS患者外周血T淋巴细胞分泌Th1细胞因子;调节ACS患者外周血CD4+CD28-和CD4+CD25+T淋巴细胞比例,使T淋巴细胞亚组趋于平衡;使得ACS患者T淋巴细胞表面TCRVβ亚家族寡克隆表达数量减少并趋于多克隆表达,通过调控抗原呈递达到抗炎和免疫调节作用,用于防治AS的发生发展具有广阔的前景。
Atherosclerosis(AS)is the main pathological process of coronary heart disease(CHD),cerebrovascular accident and peripheral vascular disease.In this century,it is presently one of the most harmful human diseases with higheat morbidity.In order to conquer this disease,many medical groups have paid attention to a series of research on the aspects of pathology,epidemiology and etiology,but the main mechanism of AS is still unknown.
Take a wide view on the study process of AS,it has experienced three stages from vascular repair to nonspecific and specific immunity.In the early of 20 century, AS was regarded as excess lipidoses on the arterial canal.To the 70's,Russell Ross found out the reparative process and chronic inflammation might be the main point. The damage of oxLDL on the endothelium could induce the activity of nonspecific immunity system.And a lot of leucocytes and macrophages were stimulated to migrate through the endarterium,swallowed oxLDL and overloaded to die.The toxic lipid was released to stimulate more cells.It has been generally proved that AS is the process of chronic inflammation.The artery is similar to the battlefield,toxic substances,such as oxLDL,act as attacker,and the defense is nonspecific immunity system.In the 90's,Erling Falk,Aarhus and Micheal David almost proposed that the rapid progress of AS was highly correlative with the unstability of plaque in the same time,they also indicated that unstable plaque may be comprised of more lipoids,inflammation and dead cells.Inflammatory cells gathered in the plaque,and released a great quantity of inflammatory factors and active substances.This would lead to cell necrosis and matrix degradation,and then the plaque became more unstable.Thrombosis would form and block up the blood supply,if the plaque was ruptured.And this was regarded as acute coronary syndrome,a clinic syndrome defined with the characteristic of unstable plaque and clinical manifestation of unstable angina and myocardial infarction.It has been found that neutrophilic leukocytes in the peripheral blood are increased and the serum level of TNF-α,IL-6 and CRP are also elevated in the ACS patients.Furthermore,K.Hansson found that activated T cells existed in the plaque,even in the peripheral blood were also detected.More interest was paid on the relationship between the specific immunity system and AS.
The specific immunity is more specific and exact than the nonspecific immunity. The nonspecific immunity assaults with all of the material out of our own,but monoclonal T cells just recognize one specific antigen.Hundreds of millions kinds of T cells are flowing with blood and dwelling in the lymphatic organ.Specific antigen could be recognize and extracted by antigen presenting cells(APC),such as macrophagocytes and dendritic cells et al.When specific T cell clone accepts the signal from antigen presenting cells,it will be activated and differentiate.The specific T cell clone could differentiate into Th cell which can induce immune responses and Ts cell which lead to immunodepressive.The exist of activated T cells in the plaque and peripheral blood suggests that immunologic mechanism may play the important role in the genesis and development of AS.The relationship between the immune system and AS is father-child relation or brother relation.And the exist of activated specific T cells clone become the core element of the question whether AS finally belong to specific immune or nonspecific immune.
T cells in the peripheral blood could be divided into different kinds of subgroups according to their phenotype and function.Usually two subgroups of CD4+ T cell and CD8+ T cell is firstly be separated on their phenotype of CD expression.And T cells can also be divided into three subgroups of help T cell(Th), cytotoxic T cell(Tc)and suppressor T cell(Ts)on their different functions.CD4+ Th cells can be classified into three subgroups of Th0,Th1 and Th2 according to the cytokine secreted.The native T cells,I.e.Th0 cell,can produce many kinds of cytokine soon after stimulated by antigen.And then Th0 cell will differentiate to Th1 and Th2 cell according to the different influence of cytokines,antigen characteristic and other hormones.Th1 cells could synthesize some cytokines such as IL-2,IFN-γand TNF-α,and promote the cellular immune.Although Th2 cells could enhance the antibody mediated humoral immunity by the secretion of cytokines of IL-4 and IL-10.In the normal circumstance,Th1/Th2 cells keep the voluntary balance to maintain proper functions.But in the pathological conditions,the balance may be broken down,the disequilibrium of subgroups possibly lead to immune disorder.In order to illuminate specific antigen activated T cells clone expands in AS,we should firstly detect whether the unbalance of T cells subgroups exist or not in the peripheral blood.
T cell receptor(TCR)is the receptor of the specific antigen on the surface of T cell.The main function of TCR is antigen recognition.The beginning of immune response was based on the TCR on the CD4+ T cell to recognize the MHC-antigen compounds on the APC surface.TCR has always been proven to be the core point of the research on the stimulation of T cells.TCR is a dimeride structure consisted ofα/βorγ/δsubunit.Two peptide chains connected with disulfide bond form transmembrance polypeptides.In our peripheral bloodα/βpeptide chains is expressed by most of T cells(95%),soα/βTCR is the main antigen recognized receptor on the mature T cells,α/βTCR can distinguish the specific antigen-MHC compounds,combine with noncovalent interaction,and the stimulated signal conducts into cellular in the last.TCR consists with three complementary determining region(CDR).Among them,CDR3 is just the binding site with the antigen determinant.And CDR3 is the hypervariable region of TCR adapted with all kinds of antigen outside.That to say,different kinds of CDR3 sequences maybe represent different T cell clone.The study on the sequence of CDR3 has already proven to be the well tools reflecting the function of T cells.In order to find the expands of specific T cell clone corresponded with unknown disease,we should detect the sequence of CDR3 in the PBTC from ACS patients.
Although until now the mechanism of AS is still unknown,it is too difficult in the fight with CHD.Scientists have tried their best to find the methods and drug to prevent and control the process of AS for the past century.Three main progresses were experienced in the treatment of acute myocardial infarction.The establishment of institution of cardiac intensive care and the treatment of intravenous thrombolytic therapy change the CHD treatment from conservative therapy to active therapy to pursue the best result for more survival myocardial muscle.It has been believed that the active therapy not only turns down the mortality of AMI,but also decreases the incidence of ischemia heart disease and refractory heart failure.In the past 20 years,the main noticeable improvements are coronary intervention treatment and intensive lipid lowing therapy.The intervention treatment eliminates the blockage of plaque,change the narrow situation and recover the blood perfusion. And intensive lipid lowing therapy could stabilize the plaque and control the process of AS unless lowing the hyperlipemia.But the mechanism of pleiotropic effect of statins is still unknown.Some study found that statins may have the potential capability of immunological regulation.The inflammatory reaction is the main cause of unstable plaque,and then statins could stabilize the plaque.We presume that statins maybe control the process the unstable plaque by the function of immunoregulation.So we present a follow up research on the ACS patients after six month lowing lipid therapy to observe the effect on the immune system of statins.It is expected to prove the relationships between the specific immune and the process of AS.
In this study,we recruited 81 hospital patients from Jan 2006 to July 2006 in zhujiang hospital.All the patients were divided into five group:control group(Control),chest pain syndrome group with normal coronary angiograph(CPS), stable angina group(SAP),unstable angina group(UAP)and acute myocardial infarction group(AMI).In the first part,the level of Th1/Th2 cytokine in the peripheral serum and the culture supematant of PBTC were detected by enzyme-linked immunosorbent assay(ELISA).The ratio of CD4+CD28- and CD4+CD25- T cell subgroups were checked by flow cytometry(FCM).The main purpose was to analyze the change of the balance of Th1/Th2 subgroups and other subgroups and to find out the clue that activated T cells exist in the peripheral blood of AS patients.In the second part,the sequences of TCR Vβchain were analyzed by reverse transcription polymerase chain raction(RT-PCR)and gene scan to confirm the expand of monoclonal T cells.At last,we performed a follow up of statins treatment of six months to observe the change of items above,and affirmed the immuno- regulation of statins.We got the result as follows:
1.The balance of T cell subgroups were broken in the peripheral blood of ACS patients
1.1 Baseline clinical characteristics in patients with CHD and healthy control subjects
All the 81 patients recruited were separated into AMI group(n=20),UAP group(n=20),SAP group(n=20),CPS group(n=11)and Control group(n=10).There is no significant difference in the baseline clinical characteristics in all of the groups(P＜0.05),such as:age;gender;smoking;the ratio of hypertension and diabetes mellitus;the serum level of total cholesterol(TC),triglyceride(TG),HDL and LDL,et al.
1.2 The serum level of Th1/Th2 cytokines in all of groups
The serum level of IL-4 in all groups was detected as 5.31±1.4,5.91±1.02, 5.7±1.4,5.47±1.1 and 6.36±1.38pg/ml,IL-10 was 32.894±9.3,33.16±9.65,32.34±9.44, 34.5±11.15 and 35.24±10.24 pg/ml.There is no significant difference among all of the groups(P＞0.05).The serum level of IFN-γin control,CPS,SAP,UAP and AMI groups was detected as 5.664±2.17,6.354±1.68,6.564±1.50,15.714±2.68 and 14.274±3.25 pg/ml and TNF-αwas 34.24±6.98,38.4±7.81,36.64±7.56,56.54±9.58 and 55.7±10.24 pg/ml.The level of IFN-γand TNF-αwere significant elevated in UAP and AMI groups than other groups(P＜0.001).
1.3 The level of Th1/Th2 cytokines in the culture supematants of PBTC of all groups
The level of IL-4 in culture supernatants of PBTC was detected as 60.94±8.7, 63.1±10.1,62.64±9.7,63.14±9.8 and 64.2±10.0 pg/ml,IL-10 was 30.14±6.15,33.7±6.24, 32.54±7.21,32.954±7.92 and 35.2±8.1pg/ml.There is no significant difference among all of the groups(P＞0.05).The level of IFN-γin culture supernatants of PBTC in control,CPS,SAP,UAP and AMI groups was detected as 1003.954±230.5, 991.84±197.2,965.5±250.9,1756.14±306.5 and 1834.44±314.5 pg/ml and TNF-αwas 476.34±98.1,486.74±115.2,482.24±125.0,861.74±172.3 and 858.74±189.4 pg/ml.The level of IFN-γand TNF-αwere significant elevated in the culture supematants of PBTC of UAP and AMI groups than other groups(P＜0.001).
1.4 The ratio of CD4+CD28- and CD4+CD25- T cell subgroups in all of the groups
The ratio of CD4+CD28- / CD4+ T cells in control,CPS,SAP,UAP and AMI groups was 2.68±0.75%,2.79±0.49%,2.68±1.71%,10.74±5.3%and 10.94±5.97%; the ratio of CD4+CD25+/CD4+ was respectively 10.29±3.71%,10.2±3.79%, 11.4±4.27%,6.43±2.68%and 5.9±2.23%.The ratio of CD4+CD28- T cell subgroups was significant increased in the peripheral blood of ACS groups;and the ratio of CD4+CD25+ T cell subgroups was decreased in ACS groups.
2.The expression of monoclonal TCR Vβsubfamilies in ACS groups
There are more than one monoclonal TCR Vβsubfamilies which can be detected in 17 cases of ACS groups;but in only three patients of SAP groups.All of the TCR Vβsubfamilies were expressed in the polyclonal way in the patients of Normal and CPS groups.In all the 20 cases of monoclonal expression,the number of patients with the expression of Vβ3,Vβ12,Vβ14,Vβ17 and Vβ22 subfamilies were more than 25%of all the cases,and were respectively 6cases,5cases,6cases, 7cases and 5cases;the number of patients with expression of Vβ6.2,Vβ7,Vβ9,Vβ18 and Vβ21 subfamilies were less than 25%,and were repectively 3cases, 3cases,2cases,1eases and 3cases.The numbers of patients with monoclonal subfamilies in ACS group were more than in other groups significantly.
3 The immunoregulation function of atorvastatin on the six month follow up
3.1 Effect of therapy of atorvastatin on patients with CAD after six month follow up
After six months follow up,the serum level of TC in AMI,UAP and SAP groups was 3.89±0.26,3.98±0.35 and 3.01±0.32mmol/L,was decreased in 31.4±10.8%,22.8±7.3%and 35.3±18.8%,respectively.The serum level of LDL was 2.25±0.35,2.064±0.26 and 1.85±0.34mmol/L,and was decrease in 36.2±17.7%,37.9±11.8%and 37.6±18.9%.The level of TG and HDL were not changed remarkable. Only 1 patient in AMI groups was found with instent restenosis.There are no discrepancy in incidence rate of MACE in all groups followed up.
3.2 Effect of atorvastatin on the serum level of Th1/Th2 cytokines in all of groups
The serum level of IL-4 in SAP,UAP and AMI groups was detected as 5.15±1.72,5.88±1.07 and 6.38±1.12pg/ml,IL-10 was为35.44±6.03,32.4±5.29 and 35.4±6.0 pg/ml.There is no significant difference compared with six months before (P＞0.05).The serum level of IFN-γin SAP,UAP and AMI groups was detected as为6.07±1.04,6.75±1.02 and 6.89±1.74 pg/ml and TNF-αwas38.2±6.79,39.5±7.38 and 40.5±7.46 pg/ml.The level of IFN-γand TNF-αwere significant decrease in UAP and AMI groups compared with six months before(P＜0.001).
3.3 Effect of atorvastatin on the level of Th1/Th2 cytokines in the culture supematants of PBTC of all groups
The level of IL-4 in culture supernatants of PBTC in control,SAP,UAP and AMI groups was detected as 67.37±5.17,61.8±14.02,66.39±14.14 and 66.95±16.06 pg/ml,IL-10 was 32.5±6.81,35.3±8.2,36.3±7.94 and 39.76±8.93pg/ml.There is no significant difference among all of the groups compared with six month before (P＞0.05).The level of IFN-γin culture supematants of PBTC in control,SAP,UAP and AMI groups was detected as 1042.1±253.1,947.47±142.6,1136.4±211.7 and 1023.6±186.7pg/ml and TNF-αwas 459.7±131.6,490.88±127.3,511.74±132.8 and 519.8±159.5pg/ml.The level of IFN-γand TNF-αwere significant decreased in the culture supematants of PBTC of UAP and AMI groups compared with six months before(P＜0.001).
3.4 Effect of storvastatin on the ratio of CD4+CD28- and CD4+CD25- T cell subgroups in all of the groups
The ratio of CD4+CD28- / CD4+ T cells in control,SAP,UAP and AMI groups was 2.6±0.5%,3.7±0.94%,3.2±1.28%and 3.29±0.9%;the ratio of CD4+CD25+/CD4+ was respectively 10.05±4.95%,10.4±2.12%,10.57±2.12%and 10.24±2.03%.The ratio of CD4+CD28- T cell subgroups of ACS groups was significant decreased in the peripheral blood and the ratio of CD4+CD25+ T cell subgroups was increased compared with six months before.
3.5 Effect of storvastatin on The expression of monoclonal TCR Vβsubfamilies in ACS groups
Only 9 cases in all 17 patients of ACS groups with monoclonal expressions before were detected the same subfamilies expression after six months follow up,the remain 8 cases were in the polyclonal expression's way.In SAP group,2 cases of all the three patients with monoclonal expressions before also can detected the same subfamilies expression.The number of patients of monoclonal expression in the ACS and SAP group were comparable(P=0.3484).
Through experiments of the above three parts,we can make the following conclusions:
①The serum level of Th1/Th2 cytokines was changed in ACS patients,immune response may be conrespond with the pathological machenism of AS;
②The level of T Th1/Th2 cytokines in the culture supematants of PBTC in ACS patients was in the unbalance way,Th1 cell was in the active condition in ACS patients;
③The ratio of CD4+CD28- and CD4+CD25+ T cell subgroups were changed in ACS patients,the active T cell and the unbalance in the proliferation of T cell subgroups may play the important role in the formation of unstable plaque;
④The expression of the monoclonal subfamilies of TCR Vβwere increased in ACS patients,the proliferation of monoclonal T cell increase revealed that antigen, presentation was more relatively with the unstable plaque.
⑤Statins can intervene the proliferation of monoclonal T cells,the secretion of Th1 CKs,and the subgroups balance.These effects provide a theorical foundation for using the statin to act as an immune inhibitor,and have the effects of regulating immunity and anti-inflammation.Thus,the foreground of using statins to prevent and cure AS is quite vast.
引文
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