树突状细胞在兔动脉粥样硬化模型中作用的研究
摘要
冠心病(coronary heart disease,CHD)是进入二十世纪以来,人类健康和生命的最大威胁。但是,其发病机制尚不明确,成为阻碍其治疗的重大障碍;针对其发病机制的研究,一直是心脏医学的热点。动脉粥样硬化(atherosclerosis,AS)是心脑血管疾病的病理病变基础。在其发展过程中,经历脂纹、脂斑、纤维斑块、粥样硬化斑块等进展时期;通过对临床发病致死病例的尸体解剖,人们最初认识AS斑块,是终末期溃疡样的动脉内膜瘤样增生;进而在非冠心病死亡的年轻病例的动脉血管壁上检测到起始阶段脂纹和脂斑的存在;最后通过对AS实验动物和临床病理的进一步观察,人们得出AS具有缓慢和逐渐进展的特点。近年来临床观察发现,冠心病终末期具有加速进展的临床表现,表现为发病突然、风险度高等特点。如果救治不及时,死亡率和致残率明显升高。分析其病理改变发现以易损斑块为主要特征,斑块不稳定到破裂,发展迅速并引起瀑布样血栓形成,导致疾病的快速和剧烈进展。人们再次认识到冠心病终末期具有加速演变和剧烈进展的特点,将这一类加速进展阶段的末期情况归纳为急性冠脉综合症(acute coronary syndrome,ACS)。冠心病在发病和进展时期的隐蔽性大,而进入终末期的致死率和致残率明显升高,对人们生活和工作构成极大的损害。如何早期发现、早期防治以及有效的减少其发病率和致残率,是医学界的热点问题。为了更好的解决这些临床问题,对其的发病机制和进展期恶化因素的研究成为高度关注的核心。
因为AS的病变过程漫长,而终末期又具有进展迅速的特点。人们发现其发病过程与创伤、肿瘤和炎症及自身免疫性疾病都具有一定的相似点,使得对AS的发病和进展机制有了种种不同的推测,类创伤、类肿瘤以及类炎症和自身免疫性疾病等等。然而,如果是创伤修复,为何越修复越进展;如果是肿瘤,为何越生长细胞调亡反而增多;如果是慢性炎症和自身免疫性疾病,又没有公认的明确的抗原来源。各种学说都在解释AS的发生发展过程中都遇到一定的困难。如何寻找突破口,最终阐明AS发病机制成为困扰各个学说的主要问题。近期,人们对ACS患者体内活化免疫细胞和细胞因子的检测以及对AS各个时期病变中活化免疫细胞检测中,发现冠心病终末期患者不仅仅在斑块病变局部可以检测到大量活化T淋巴细胞和单核细胞,其外周血中也可以检测到前炎症因子的高水平表达,提示在AS的终末阶段,出现全身炎症反应增强的表现,又根据活化T淋巴细胞的出现,使得人们对炎症和免疫参与疾病发展的关注逐渐升温。针对ACS患者外周血和病变组织中活化T淋巴细胞亚组、分型、受体及黏附附因子等方面的研究都发现病变局部有大量活化T淋巴细胞存在,并出现亚组分型和蛋白表达各方面的差异。斑块内T淋巴细胞活化的根源在那里,使得人们自然注意到作为诱导T淋巴细胞活化的主要抗原呈递细胞——树突状细胞(dendritic cells,DCs)的存在。斑块内是否存在作为抗原提成的DCs;DCs的来源如何:DCs是否为成熟状态,其功能状况怎样;DCs是否吞噬抗原并将抗原肽刺激信号提呈给初始T淋巴细胞,而诱导T淋巴细胞增殖和活化等等问题,成为关注的热点。
作为DCs的研究,因为DCs在体内各组织中存在细胞量少,在外周血中DCs占有核细胞的比例低于1%,在正常血管壁内DCs更难以检测,这些给DCs的研究带来一定的困难。部分学者通过培养外周血单个核细胞(peripheral bloodmononuclear cell,PBMC)来源的DCs,在体外获得一定数量的DCs,并对来源于AS不同阶段患者的DCs成熟状态及免疫功能作比较,发现ACS患者PBMC来源的DCs高表达CD80、CD86和CD83等共刺激分子和黏附分子,表现为成熟状态,并且具有较强的活化T淋巴细胞的能力。提示ACS患者体内可能存在诱导DCs活化的物质,使得DCs表现为成熟阶段。而应用氧化低密度脂蛋白(oxidatedlow density lipoprotein,oxLDL)、尼古丁和热休克蛋白(heat shock protein,HSP)等AS相关物质都能在体外诱导DCs成熟,并具有一定的免疫活性。但是,对于DCs的研究如果始终停留在对可能诱导AS的抗原刺激物对DC活化作用的检测,或者对ACS患者PBMC体外培养DCs成熟标准的检测,则对AS的形成和发展机制无进一步的深入。
为了突破目前研究的局限性,进一步证实DCs确实参与了AS的进展,并在ACS患者不稳定斑块的形成中起到启动和活化T淋巴细胞,而使得炎症反应增强的作用。根据DCs在体内经历从骨髓干细胞发育成DCs前体细胞,到经过血液迁徙到抗原位点,未成熟细胞通过吞噬抗原,并提呈抗原成为成熟细胞,后再迁徙到淋巴结T细胞区激活初始T淋巴细胞增殖全过程的情况。我们设想可以通过选择合适的AS动物模型,分析AS动物模型中典型斑块与炎症的关系,进而分析斑块局部炎症与DCs分布与功能变化情况,以验证外周血DCs分布情况从侧面反应其迁徙到血管壁内的状况,并通过体外实验观察AS的可能危险因素对DCs成熟及免疫活化的影响。如果实验动物体内存在诱导DCs活化的因素,进而导致斑块内DCs聚集和活化,吞噬可能存在的抗原物质而活化,其血管壁内的DCs分布必然会上升,诱导炎症反应增强,从而使得斑块活化及不稳定。因此,本实验研究从选择合适动脉硬化动物模型出发,以期在合适的动脉斑块中检测炎症及DCs成熟状况,并检测AS动物模型中可能存在的危险因素与DCs成熟的关系,探讨DCs在易损斑块形成过程中的作用,为冠心病免疫发病机制的阐明奠定一定实验基础。
本课题分为三个部分,首先以兔腹主动脉粥样硬化模型为研究对象,应用病理切片、血管造影及体外超声检测斑块成膜情况,分析其斑块性质,建立研究模型基础;从建立的AS实验动物出发,应用流式细胞技术和免疫组织化学观察DCs在血液、血管组织、脾脏和肝脏及淋巴结的分布情况,重点观察DCs的分布情况对AS发生发展的影响;体外培养实验动物骨髓源DCs,并通过ELISA、流式细胞技术及免疫检测技术观察模型动物的自体血清与ox-LDL刺激对DCs成熟诱导情况,以明确AS动物模型中DCs与典型斑块形成的可能机制。
结果分述如下。
1.新西兰兔的模型制备及相关检测
1.1动脉粥样硬化新西兰兔成模情况及各组兔一般情况的比较
16只雄性纯种新西兰白兔,体重2.50±0.14kg,随机分为对照组和实验组,各组予以腹主动脉内膜的球囊损伤后,实验组以高脂饮食喂养2月,对照组则以正常饮食喂养2月。给予高胆固醇饮食喂养2月,喂养后兔的体重较喂养前在实验组与对照组均数均明显增加,差异有显著性意义(P=0.000);实验组喂养后较喂养前的TC、TG、LDL均数增高,差异有显著性意义(P=0.000);实验组喂养后与对照组喂养后比较,其TC、TG、LDL均数明显增高,差异有显著性意义(P=-0.000)。
1.2各组新西兰白兔腹主动脉造影及外周血管超声情况的比较
两组新西兰白兔喂养2月后,行腹主动脉造影检查及外周血管超声检查,分别比较喂养2月后两组新西兰白兔腹主动脉硬化斑块形成对动脉管腔变化的影响,并观察麦角新碱对球囊剥脱部位诱发痉挛情况的比较。
在腹主动脉造影下,测量药物刺激后血管收缩率,t检验显示实验组的损伤部位和未损伤部位之间以及实验组的损伤部位与对照组的损伤部位之间血管收缩率差异存在显著性意义(P=0.000)。
以体表超声评价血管偏心指数(斑块偏心指数=斑块最厚处斑块厚度/该部位对侧斑块最薄处斑块厚度)。结果显示偏心指数较大的斑块在药物刺激后血管收缩明显增加,偏心指数与血管收缩率之间呈显著正相关(r=0.983,P=0.000)。
1.3各组新西兰白兔腹主动脉内膜增生情况比较:
两组新西兰白兔喂养2月后,分别留取球囊剥脱部位及临近剥脱部位的腹主动脉血管标本。比较喂养2月两组新西兰白兔主动脉形态学,实验组光镜下见中膜和内膜均明显增厚,有较多增生的血管平滑肌细胞,呈环形排列整齐,新生内膜中有大量细胞外基质形成,管腔明显狭窄。各种参数如内弹力膜环绕总面积、新生内膜面积、管腔面积及内膜增生百分比的结果比较,四组之间明显差异,独立样本t检验发现实验组球囊剥脱部位与临近剥脱部位和对照组相比,新生内膜面积显著增大,差异有显著性意义(P=0.004),管腔面积显著减小,差异有显著性意义(P=0.000),内膜增生百分比明显增大,差异有显著性意义(P=0.001)。说明实验组新西兰白兔球囊剥脱部位的主动脉内膜有新生内膜形成。
1.4各组新西兰白兔血清NO水平及血管标本内eNOS酶蛋白表达的比较
采用硝酸还原酶法对两组新西兰白兔喂养2月后的血清NO水平进行检测。结果显示实验组与对照组比较,血清NO水平下降的差异有显著性意义(P=0.002)。
采用免疫组化对各组血管组织中eNOS酶蛋白含量和定位进行检测,结果显示对照组血管组织中,内皮细胞有阳性着色,球囊剥脱部位与临近剥脱部位内皮细胞阳性着色点无明显差异;实验组与对照组相比球囊剥脱部位内膜的阳性着色减少,内皮下层出现散在的阳性着色点,而临近球囊剥脱部位内膜阳性着色点较对照组无明显差异。说明高脂血症可以导致血浆NO水平下降,但对血管内皮eNOS酶表达无明显影响,只有在血管内皮损伤后,血浆胆固醇进入内膜进一步演变成有害胆固醇蛋白,进而对内皮功能产生影响。
2.兔动脉粥样硬化与炎症及细胞免疫的关系
通过ELISA方法检测实验组血浆炎症因子表达水平,并流式细胞检测技术和免疫组织化学方法,分析AS动物模型血管壁内DCs及免疫细胞的分布,观察是否存在与正常对照组之间的区别。
2.1血生化指标及炎症指标的检测:
制备模型2月后,通过ELISA方法检测实验动物血浆炎症因子表达水平,实验组兔血清sICAM、sVCAM、IL-6、TNF、hsCRP的含量与对照组相比,存在显著性差异(P＜0.01)。提示在兔动脉粥样硬化的发生、发展过程中存在炎症反应,黏附分子与CRP等炎症因子共同促进动脉粥样硬化的发展。
2.2兔DCs表型分子S100及抗原呈递细胞活性分子CD86、MHCⅡ分别在两组剥脱及未剥脱血管部位的表达情况
制备模型2月后,经流式细胞术检测,实验组血管剥脱部位的CD86、MHCⅡ、S100含量分别与未剥脱部位、对照组剥脱部位的血管相比明显升高,差异有显著意义(P＜0.01);CD86、MHCⅡ、S100的含量在实验组未剥脱部位、对照组剥脱部位及未剥脱部位的血管两两之间,差异无显著性意义(P＞0.05)。在模型兔剥脱部位血管壁组织内检测到兔特异性的DCs标记分子明显增多,说明实验组较对照兔腹主动脉血管壁内DCs明显升高;而检测抗原呈递细胞活性分子CD86及MHCⅡ含量也较对照组明显升高,说明抗原呈递细胞活化与AS的进展高度相关
3.兔外周血来源的DCs可以在模型自体血清及AS相关物质刺激下诱导成熟,并具有诱导T细胞增殖的能力
制模前兔外周血来源的DCs分为4组,空白组予以PBS干预,对照组予以制模前的兔自体血清干预(10%),实验组予以制模后兔自体血清干预(10%),ox-LDL组予以ox-LDL(10μg/mL)干预。
3.1兔外周血源的DCs培养及形态学鉴定:
兔外周血源的DCs在光学显微镜下可见悬浮生长,部分细胞聚集成簇,细胞表面有数量不等、形态不一的树突样突起;在扫描电镜下细胞呈不规则形,表面粗糙,胞体有形态不一的突起。
3.2各组兔外周血源的DCs的表型流式细胞学检测:
空白组、实验组、对照组以及ox-LDL组的兔外周血源的细胞培养前后的表型流式细胞学检测结果经One-Way ANOVA检验显示:MHCⅡ(P=0.000)、CD86(P=0.001)、CD14(P=0.000)在各自的不同组间的差异有显著性意义,对照组、实验组与ox-LDL组的MHCⅡ、CD86的表达较空白组上调,差异有显著性意义(P<0.05),实验组与ox-LDL组的CD14表达下调明显,差异有显著性意义(P<0.01)。对照组表现为CD86~+/MHCⅡ~+/CD14~(dim),区别于空白组单核/巨噬细胞的MHCⅡ~+/CD14~+的表型特征。在实验组自体血清环境以及ox-LDL组的ox-LDL刺激后,MHCⅡ和CD86表达继续上调,CD14~+/aim则转化为CD14~-,其MHCⅡ~+/CD86~+/CD14~-的表达为成熟树突状细胞的表现。
3.3混合淋巴细胞反应:
各组的兔细胞按照不同比例(1:10、1:50、1:100)与同种异源的T淋巴细胞混合,加DMSO后用酶标仪检测波长490nm的A值。结果显示各组均可产生不同程度的增殖反应,析因分析表明混合比例因素(P=0.000)和分组因素(P=0.000)对MLR的结果影响均有显著性意义,且1:10的比例时增殖反应最强。One-Way ANOVA检验各组在不同混合比例下的差异,实验组(P=0.026)各比例间差异有显著性意义。One-Way ANOVA检验不同比例的MLR在各组之间的差异,结果提示在1:10及1:50混合比例时各组之间比较有显著性意义(P=0.002),且ox-LDL组和实验组较空白组具有更强的刺激异体淋巴细胞增殖的能力,比较有显著性意义(P<0.05),说明经高脂血清环境培养的DCs与ox-LDL刺激生成的DCs具有更强的抗原提呈能力,并且随着DCs数量的增多,刺激同种异体淋巴细胞增殖的能力增强。
通过上述三个部分的实验,我们得出以下结论:
1通过球囊内皮剥脱并高脂饮食2月后,可形成明确的兔腹主动脉硬化斑块,该斑块具有显著的内膜增生、管腔狭窄等特点;
2模型兔腹主动脉斑块具有部分类似人动脉硬化的特点,体表超声显示该斑块呈密度低、偏心形等特点,麦角新碱诱发下腹主动脉造影显示球囊内皮剥脱部位可诱发明显的血管痉挛;
3斑块形成部位eNOS减少说明斑块部位内皮功能严重受损;
4模型兔血浆炎症因子分泌增多,说明炎症与AS形成高度相关;
5模型兔血管壁DCs的比例增多,且其活性表型分子表达增多提示DCs可能参与AS的形成;
6外周血分离的PBMC可在IL-4及GM-CSF的联合诱导下,培养出形态及表型稳定的DCs;
7模型兔自体血清可诱导DCs的成熟及促进T淋巴细胞的增殖,可能参与AS形成;
8 ox-LDL同样可诱导DCs的成熟及免疫活性,可能在本实验动物模型中占据主要的抗原成分地位。
Coronary heart disease(CHD)has been the greatest threaten to human beings since the arrival of the 21th century.However,the pathogenesis of CHD has not been clearly identified,so it is a great hindrance to the treatment of CHD.Thus,to study on the pathogenesis of CHD has become a pop trend of cardiology.It is generally accepted that atherosclerosis(AS)is the pathologic basis of cardiac cerebro vascular disease.AS is considered to be a progressing course which including the formation of fatty streak,fatty plaque,fibrous plaque,atherosclerotic plaque,and so on.People's initial recognition on AS is the anabrotic tumor-like proliferation of artery endomembrane in the final stage by clinical autopsy of dead cases.Subsequently, people discovered fatty streak and fatty plaque on arterial vessel walls in young cases which died of any disease but CHD.Finally,people got to know that AS proceeds slowly and gradually by making animal experiments and further clinical observations. Clinical observations in recent years had found that end stage CHD would have an accelerated progress which appears as sudden onset of an event with a high rate of risk,and so on.The mortality and mutilation rate will be surprising high if the salvages and treatments are not available in time.The predominant pathogenic characteristic of the arteries in these cases is the forming of unstable plaques.Plaques in this situation are always fragile so they may disrupt easily and quickly.Eventually, a chute-like thrombogenesis which will worsen the disease is caused.People again realized that end stage CHD may evolve acceleratedly and progress intensively.So we call this kind of end stage CHD acute coronary syndrome(ACS).The concept is emphasizing on a worsen stage in the developmental course of CHD.The clinical symptoms and signs are larvaceous in the initial and advancing stages of CHD,but when it comes to the end stage,the mortality and mutilation rate is getting surprisingly high,making great damage or loss to people' daily life or work.
AS experiences a slow and continuous pathological change,but may speed up in the end stage of its natural course.People found that pathological process of this kind has some similarity to that of trauma,tumor,inflammation,or autoimmune diseases, and so on.So different kinds of presumes on the morbility and progressing mechanisms of AS emerged,such as theories of trauma-like disease,tumor-like disease,inflammation-like disease,or autoimmune disease,just name a few.However, no even a literature documents that AS is a process of repair in any kind of trauma for that AS advances to a more serious stage when more repairs are done.No one confirms that AS is a kind of tumor for that more cells will experience apoptosis with more cellular proliferation.No one had even found out an antigen that would generally be accepted if it is proved to be a chronic inflammation or an autoimmune disease.Any of these theory faces a difficulty in explaining the morbility and advancing mechanism of AS.So how to make a breakthrough to find out a more convincing theory to help the explanation of AS pathogenesis is a difficulty to any one of the existed theory.Detections on the activated immunocells and cytokines in vivo in ACS cases and,detections made recently on the activated immunocytes in different stages of AS indicate that not only can we detected a great sum of activated T cell and monocytes,but also we can detected a high level expression of pro-inflammatory factors,in the peripheral blood,this suggests that a intensified inflammatory reaction in the body.With more knowledge on activated T cells are obtained,people pay more attention to the roles of inflammation and immunity in the development of AS than ever before.Studies on T cell subgroup,T cell typing, receptor expression and adhension factor had found that a great sum of activated T cells appeared in the peripheral blood and affected tissues of ACS cases.The difference in subgroups,typing and protein expression can also be noticed.The desire to know what activate T cells makes people pay more and more attention to the existence of dendritic cells(DCs).DC is a main kind of antigen presenting cell(APC) which are capable to induce the activation of T cells.Questions as whether DCs exist in the AS plaques or not,what is the origin of DCs,whethr DCs are in a mature status or not and what are their functioning status,whether DCs phagocytize the antigens and then present the antigen peptide-stimulated signs to incipient T cells so as to induce the proliferation and activation of T cells,have becoming the focus presently.
Normally speaking,DCs can hardly be seen in all kinds of body tissues.DCs are less than 1%of all the karyocytes in peripheral blood.It is even harder to find out any DCs in the normal vessel walls.All of these make it difficult to make a further study on DCs.Some scholars obtain DCs in vitro by cultivation of mononuclear cells from peripheral blood.They compared the maturity and immune functions of DCs in different stages of AS.They found that the PBMC derived DCs in ACS cases showed a high expression of costimulatory molecules and adhension molecules such as CD80, CD86,CD83,and so on.What's more,they had found that the DCs appeared to be mature and had a strong ability to activate T cells.All of these suggest that some substances which are capable to induce the activation of DCs and make them appear to be mature,exist in ACS cases.AS-related substances such as oxidated low density lipoprotein,nicotine and heat shock proteins,may induce the maturity of DCs and, give them the immune activity.However,if the study just retents on the in vitro experiments which may only confirm that the AS-induced antigen stimulus play roles in the activation of cells,or only makes a detection on the maturity standards of PBMC derived DCs cultured in vitro in ACS cases,no further progress will be made on the forming and advancing mechanism of AS.So,the questions mentioned above still can't be confirmed.
In order to further confirm that DCs certainly play a role in the progress of AS, and that they initiate and activate T cells in the forming of unstable plaques in ACS cases so as to intensify the inflammatory reaction and,on the basis that DCs may experience a developmental process of transforming from bone marrow stem cells to DCs precursor cells in vivo firstly,and then they will deadexis to the antigen-located sites,then the immature DCs may phagocytize and present the antigens so as to become mature cells which will deadexis to the lymph node T cell regions subsequently to activate incipient T cells for their further proliferation.We make a hypothesis that for verifing the distribution of DCs in peripheral blood and reflection its movement from side face into the vessel wall,and investigating the affection of the possible risk factors of AS for the maturation of the DCs and immune activation in vitro,we should analyse the relationship between typical plaque and inflammation in animal model which is suitable for researching the AS,and further to analyse local inflammation of plaque and the distribution and function of DCs.If DCs aggregate and be activated by phagocytizing antigens in the plaques in ACS cases, their distribution levels in vessel walls may raise,so a redistribution of DCs in peripheral blood and lymphs can be noticed.We started our study with the presume that detection of DCs proportion and quantity in different stages and different tissues in their genesis and developing courses would help to investigate the roles of DCs in the forming of unstable plaques in AS,so it may help to set up a experimental ground for the establishment of a confirmed immune mechanism of CHD.
Our experiments can be divided into three portions.First of all,to set up basic research model,we should take the rabbit model abdominal of aorta atherosclerosis as the objection,and apply the pathology slice,angiography and vitro ultrasound for detection the plaque formation and analysis the nature of the plaque;the distribution of DCs in the blood,vascular tissue,spleen and liver and lymph nodes and stressly the impact of the distribution of DCs on the generation and development of AS should be investigated by flow cytometry and immunohistochemistry from setting up the laboratory animals.For elucidating the possible mechanism of the formation of plaque,cuture the bone marrow-derived DCs in vitro,and observation the maturation of DCs stimulated by OxLDL through ELISA,flow cytometry and ELISA should be done.The results are as follows:
1 Preparation the New Zealand rabbit model and related testing
1.1 Established status of the New Zealand rabbits model of atherosclerosis and comparison the general situation in each group.
16 pure-bred New Zealand white male,weighing 2.50±0.14kg,are randomly divided into control group and experimental group.After injuring the intima of abdominal aorta of the rabbit by balloon,high-fat diet should be fed with for 2 months in experimental group,and normal diet should be fed with in control group.After giving high-cholesterol diet for 2 months,the body weight of rabbits after feeding than before both in the experi-mental group and the control group are significantly increased,and have significant difference(P=0.000);the levels of TC, TG,LDL in the experimental group after feeding higher than before respectively, and have significant difference(P=0.000);the levels of TC,TG,LDL in the experimental group are significantly increased than in the control group respectively, and have the significant difference(P=0.000).
1.2 Comparison the result of angiography in abdominal aortic and peripheral vascular ultrasound imaging of the New Zealand white rabbits in each group.New Zealand White rabbits are fed up for 2 months,then we take the angiography in abdominal aortic and peripheral vascular ultrasound for comparing the influence of atherslerosis plaque formation on abdominal aortic lumen and the ergometrine-induced spasm in site where denudated by balloon in two groups of New Zealand White rabbits.
The drug-stimulated vascular contraction rate is measured in abdominal aortic angiography,and there have significant difference between injuried sites and uninjuried sites of the experimental group(P=0.000),and injuried sites of the experimental group and the control group(P=0.000)by t tests.
Blood vessels eccentricity index is evaluated by ultrasound in the body surface (plaque eccentricity index=plaque thickness of the thickest plaque site/plaque thickness of the contralateral site of thinnest site).The results show that a larger plaque eccentricity index have a marked increase in vasoconstriction that stimulated by drug,and eccentricity index rate positive correlated significantly with vasoconstriction(r=0.983,P=0.000).
1.3 Comparision the intimal hyperplasia of abdominal aortic of New Zealand white rabbits in each group
The specimens of the near site and the site of balloon denudation of the abdominal aortic endarterectomy should be collected in two groups after feeding up the New Zealand white rabbits for 2 months,respectively.Then the aortic morphology shoud be compared,membrane and intimal thickener significantly in experimental group in the light microscope,vascular smooth muscle cells proliferated more,and arranged neatly in ring,a large number of extracellular matrix formed in neointimal,and narrowed the lumen significantly.
There have significant difference in parameters such as around area of the internal elastic membrane,neointimal area,lumen area and the percentage of intimal hyperplasia area to lumen area among four groups,the neointimal area increased significantly higher in the site and the near site of endarterectomy by balloon in exprimental group than in the control group,and the difference is significant(P= 0.004)in independent samples t test,as well as the percentage of the intima hyperplasia(P=0.001),but lumen area decreased(P=0.000),indicating that neointimal is generated in the aorta denudation site by balloon in experimental New Zealand rabbit group.
1.4 Comparison the levels of NO in serum and eNOS protein expression in vascular specimens of New Zealand white rabbits in each group.
After feeding up New Zealand white rabbits for 2 month,the levels of serum NO in 2 group detected by nitrate reductase method.And the results showed that compared with the control group,the levels of serum NO in the experimental group have significant dicreased(P=0.002).The eNOS protein in vascular tissue are detected in quantity and location by immunohistochemistry for each group,and the results show that,endothelial cells have stained positively in vascular tissue of the control group,and the positive stained sites of endothelial cells have no significant difference in and near in balloon endarterectomy site.Compared with the control group,positive staining point of the intima in balloon endarterectomy site have reduced in the experimental goup,and the positive staining points scatter in the lower endothelial,but in the near site of balloon endarterectomy have no significant difference.It indicated that hyperlipidemia could lead to a reduction in the levels of serum NO,but have no significant effect on the expression of vascular endothelial eNOS enzyme,only in the vascular endothelial injuried,cholesterol could migrate from the serum into the intima and further evolute into harmful cholesterol protein, and thus have an influence on endothelial function.
2 The relationship between rabbit atherosclerosis and inflammation,cell immunity The existence difference between the normal control group and the experimental group are observed though detection the expression levels of inflammatory cytokines in serum by ELISA,and analysis the distribution of DCs and immune cells in the AS vessel wall by flow cytometry and immunohistochemistry.
2.1 Detection the index of blood biochemical and inflammation The levels of serum inflammatory cytokines are detected by ELISA after preparation the model for 2 months,and there have significant difference in the content of serum sICAM,sVCAM,IL-6,TNF,HsCRP between experimental group and control group(P＜0.01).It indicated that inflammatory response exist in generate and development of AS,and adhesion molecules,CRP and other inflammatory cytokines promote the development of AS.
2.2 The expression of S100 of DCs phenotype molecular and CD86,MHCII of antigen presenting moleculars in and near in vascular endarterectomy site
Detection the expression levels of MHCⅡ,CD86,S100 by flow cytometry after preparation model for 2 months,and the levels of MHCⅡ,CD86,S100 are higher in endarterectomy site in experimental group than near in endarterectomy site in experimental group,endarterectomy site in control group(P＜0.0l),but have no significant difference among near in endarterectomy site in experimental group, endarterectomy site in control group,near in endarterectomy site in control group(P＞0.05).The rabbit-specific molecular markers of DCs markedly increased in endarterectomy site in the rabbit model of vessel wall tissue,indicating that the expression of DCs are increased more in abdominal aorta vascular wall than control group.The content of CD86 and MHCII molecules of antigen-presenting cell activity moleculars are also significantly higher than the control group,indicating that activation of antigen-presenting cells is highly relevant to the development of AS.
3 The peripheral blood DCs of rabbit can be stimulated and induced to maturation by AS related substance,and have the ability to induce T cell proliferation
3.1 Culture and identify the rabbits peripheral blood derived DCs
The cultured rabbit peripheral blood cells is that suspended growth,some clustered together and different quantity and shape in dendritic-like protrusion on the surface,which can be seen under the light microscope,and irregular in shape,rough surface,different patterns of protrusion under scanning electron microscopy.
3.2 Detection the phenotype of rabbits peripheral blood derived DCs by flow cytometry in each group
The phenotype of rabbit peripheral blood cell which before and after cuture in the experimental group,the control group and the LPS group are detected by flow cytometry,and the results are tested by One-Way ANOVA and showed that:MHCⅡ(P=0.000),CD86(P=0.001),CD14(P=0.000)have significant difference between groups,respectively;compared with control group,the expression of MHCII,CD86 are upregulated in the experimental group and the ox-LDL group,and the difference is significant(P＜0.05),the expression of CD14 are significantly reduced in experimental group and ox-LDL group,and the difference is significant (P＜0.01).The phenotype of the control group is CD86~+/MHCⅡ~+/CD14~(dim),and different from monocytes/macrophages cells with MHCⅡ~+/CD14~+phenotype in the control group.Experimental group at the environment and the autologous serum ox-LDL group of ox-LDL stimulation,The expression MHCⅡand CD86 remain increased,and CD14~+/~(dim)is tranformed into CD14~-,and the phenotype of MHCⅡ~+/ CD86~+/CD14~-is the matured dendritic cells expression.
3.3 Mixed allogenic Lymphocyte reaction
According to the different proportion(l:10,1:50,1:100),each group rabbit cells are mixed with allogenic T-lymphocytes,A value is detected at 490nm wavelength by microplate reader after adding DMSO.The results show that proliferative response is vary in each group,showed that the proportion of factors(P=0.000)and group factors(P=0.000)have impact on the results of MLR by factorial analysis, and the significance is significant,and at the ratio of 1:10,the proliferative response is the strongest.There have significant difference among the different mixed ratio in the experimental group(P=0.026)by One-Way ANOVA test.Compared the defference of MLR in each group by One-Way ANOVA test,and the results suggest that at the mixed ratio of 1:10 and 1:50,the difference is significant among groups(P=0.002),and the ability to stimulate allogeneic lymphocyte to proliferate is more stronger in the ox-LDL group and the experimental group than in the control group,and the difference is significant(P＜0.05),suggesting that DCs cultured with high fat environment and stimulated by ox-LDL have more ability to present antigen, and with the increase of DCs,the ability to stimulate allogeneic lymphocyte proliferation is strengthen.
Through above three-part test,we draw the following conclusions:
1 Through endothelial denudation by balloon and high-fat diet for 2 months, rabbit abdominal atheroslerosis plaque may be the generation,and the plaque are characterized by significant intimal hyperplasia and narrow lumen;
2 The characteristics of rabbit model of abdominal aortic arteriosclerosis plaque are partly similar with people,the plaque density was low and with eccentric shape which showed by surface ultrasound,and with induced by ergonovine,the balloon-induced endothelial denudation site showed significantly of vasospasm by abdominal aortic angiography;
3 The site of plaque generation have lower eNOS,indicating that endothelial function are damaged severely;
4 The secretion of inflammatory cytokines are increased in the plasma of rabbit mode,suggesting that inflammation high associated with the generation of AS;
5 The proportion of DCs are increased in the vessel wall of rabbit model of and the molecular phenotype of DCs are activated,suggesting that DCs may be involved in the generation of AS;
6 Peripheral blood derived PBMC can be induced by IL-4 and GM-CSF into a stable morphology and phenotype of DCs;
7 Autologous serum of rabbit model can induce maturation of DCs and promote the proliferation of T lymphocytes,thereby may be involved in the generation of AS;
8 ox-LDL can induce maturation and immune activity of DCs,and probably occupy the status of the main components of the antigen in this experimental animal models.
因为AS的病变过程漫长,而终末期又具有进展迅速的特点。人们发现其发病过程与创伤、肿瘤和炎症及自身免疫性疾病都具有一定的相似点,使得对AS的发病和进展机制有了种种不同的推测,类创伤、类肿瘤以及类炎症和自身免疫性疾病等等。然而,如果是创伤修复,为何越修复越进展;如果是肿瘤,为何越生长细胞调亡反而增多;如果是慢性炎症和自身免疫性疾病,又没有公认的明确的抗原来源。各种学说都在解释AS的发生发展过程中都遇到一定的困难。如何寻找突破口,最终阐明AS发病机制成为困扰各个学说的主要问题。近期,人们对ACS患者体内活化免疫细胞和细胞因子的检测以及对AS各个时期病变中活化免疫细胞检测中,发现冠心病终末期患者不仅仅在斑块病变局部可以检测到大量活化T淋巴细胞和单核细胞,其外周血中也可以检测到前炎症因子的高水平表达,提示在AS的终末阶段,出现全身炎症反应增强的表现,又根据活化T淋巴细胞的出现,使得人们对炎症和免疫参与疾病发展的关注逐渐升温。针对ACS患者外周血和病变组织中活化T淋巴细胞亚组、分型、受体及黏附附因子等方面的研究都发现病变局部有大量活化T淋巴细胞存在,并出现亚组分型和蛋白表达各方面的差异。斑块内T淋巴细胞活化的根源在那里,使得人们自然注意到作为诱导T淋巴细胞活化的主要抗原呈递细胞——树突状细胞(dendritic cells,DCs)的存在。斑块内是否存在作为抗原提成的DCs;DCs的来源如何:DCs是否为成熟状态,其功能状况怎样;DCs是否吞噬抗原并将抗原肽刺激信号提呈给初始T淋巴细胞,而诱导T淋巴细胞增殖和活化等等问题,成为关注的热点。
作为DCs的研究,因为DCs在体内各组织中存在细胞量少,在外周血中DCs占有核细胞的比例低于1%,在正常血管壁内DCs更难以检测,这些给DCs的研究带来一定的困难。部分学者通过培养外周血单个核细胞(peripheral bloodmononuclear cell,PBMC)来源的DCs,在体外获得一定数量的DCs,并对来源于AS不同阶段患者的DCs成熟状态及免疫功能作比较,发现ACS患者PBMC来源的DCs高表达CD80、CD86和CD83等共刺激分子和黏附分子,表现为成熟状态,并且具有较强的活化T淋巴细胞的能力。提示ACS患者体内可能存在诱导DCs活化的物质,使得DCs表现为成熟阶段。而应用氧化低密度脂蛋白(oxidatedlow density lipoprotein,oxLDL)、尼古丁和热休克蛋白(heat shock protein,HSP)等AS相关物质都能在体外诱导DCs成熟,并具有一定的免疫活性。但是,对于DCs的研究如果始终停留在对可能诱导AS的抗原刺激物对DC活化作用的检测,或者对ACS患者PBMC体外培养DCs成熟标准的检测,则对AS的形成和发展机制无进一步的深入。
为了突破目前研究的局限性,进一步证实DCs确实参与了AS的进展,并在ACS患者不稳定斑块的形成中起到启动和活化T淋巴细胞,而使得炎症反应增强的作用。根据DCs在体内经历从骨髓干细胞发育成DCs前体细胞,到经过血液迁徙到抗原位点,未成熟细胞通过吞噬抗原,并提呈抗原成为成熟细胞,后再迁徙到淋巴结T细胞区激活初始T淋巴细胞增殖全过程的情况。我们设想可以通过选择合适的AS动物模型,分析AS动物模型中典型斑块与炎症的关系,进而分析斑块局部炎症与DCs分布与功能变化情况,以验证外周血DCs分布情况从侧面反应其迁徙到血管壁内的状况,并通过体外实验观察AS的可能危险因素对DCs成熟及免疫活化的影响。如果实验动物体内存在诱导DCs活化的因素,进而导致斑块内DCs聚集和活化,吞噬可能存在的抗原物质而活化,其血管壁内的DCs分布必然会上升,诱导炎症反应增强,从而使得斑块活化及不稳定。因此,本实验研究从选择合适动脉硬化动物模型出发,以期在合适的动脉斑块中检测炎症及DCs成熟状况,并检测AS动物模型中可能存在的危险因素与DCs成熟的关系,探讨DCs在易损斑块形成过程中的作用,为冠心病免疫发病机制的阐明奠定一定实验基础。
本课题分为三个部分,首先以兔腹主动脉粥样硬化模型为研究对象,应用病理切片、血管造影及体外超声检测斑块成膜情况,分析其斑块性质,建立研究模型基础;从建立的AS实验动物出发,应用流式细胞技术和免疫组织化学观察DCs在血液、血管组织、脾脏和肝脏及淋巴结的分布情况,重点观察DCs的分布情况对AS发生发展的影响;体外培养实验动物骨髓源DCs,并通过ELISA、流式细胞技术及免疫检测技术观察模型动物的自体血清与ox-LDL刺激对DCs成熟诱导情况,以明确AS动物模型中DCs与典型斑块形成的可能机制。
结果分述如下。
1.新西兰兔的模型制备及相关检测
1.1动脉粥样硬化新西兰兔成模情况及各组兔一般情况的比较
16只雄性纯种新西兰白兔,体重2.50±0.14kg,随机分为对照组和实验组,各组予以腹主动脉内膜的球囊损伤后,实验组以高脂饮食喂养2月,对照组则以正常饮食喂养2月。给予高胆固醇饮食喂养2月,喂养后兔的体重较喂养前在实验组与对照组均数均明显增加,差异有显著性意义(P=0.000);实验组喂养后较喂养前的TC、TG、LDL均数增高,差异有显著性意义(P=0.000);实验组喂养后与对照组喂养后比较,其TC、TG、LDL均数明显增高,差异有显著性意义(P=-0.000)。
1.2各组新西兰白兔腹主动脉造影及外周血管超声情况的比较
两组新西兰白兔喂养2月后,行腹主动脉造影检查及外周血管超声检查,分别比较喂养2月后两组新西兰白兔腹主动脉硬化斑块形成对动脉管腔变化的影响,并观察麦角新碱对球囊剥脱部位诱发痉挛情况的比较。
在腹主动脉造影下,测量药物刺激后血管收缩率,t检验显示实验组的损伤部位和未损伤部位之间以及实验组的损伤部位与对照组的损伤部位之间血管收缩率差异存在显著性意义(P=0.000)。
以体表超声评价血管偏心指数(斑块偏心指数=斑块最厚处斑块厚度/该部位对侧斑块最薄处斑块厚度)。结果显示偏心指数较大的斑块在药物刺激后血管收缩明显增加,偏心指数与血管收缩率之间呈显著正相关(r=0.983,P=0.000)。
1.3各组新西兰白兔腹主动脉内膜增生情况比较:
两组新西兰白兔喂养2月后,分别留取球囊剥脱部位及临近剥脱部位的腹主动脉血管标本。比较喂养2月两组新西兰白兔主动脉形态学,实验组光镜下见中膜和内膜均明显增厚,有较多增生的血管平滑肌细胞,呈环形排列整齐,新生内膜中有大量细胞外基质形成,管腔明显狭窄。各种参数如内弹力膜环绕总面积、新生内膜面积、管腔面积及内膜增生百分比的结果比较,四组之间明显差异,独立样本t检验发现实验组球囊剥脱部位与临近剥脱部位和对照组相比,新生内膜面积显著增大,差异有显著性意义(P=0.004),管腔面积显著减小,差异有显著性意义(P=0.000),内膜增生百分比明显增大,差异有显著性意义(P=0.001)。说明实验组新西兰白兔球囊剥脱部位的主动脉内膜有新生内膜形成。
1.4各组新西兰白兔血清NO水平及血管标本内eNOS酶蛋白表达的比较
采用硝酸还原酶法对两组新西兰白兔喂养2月后的血清NO水平进行检测。结果显示实验组与对照组比较,血清NO水平下降的差异有显著性意义(P=0.002)。
采用免疫组化对各组血管组织中eNOS酶蛋白含量和定位进行检测,结果显示对照组血管组织中,内皮细胞有阳性着色,球囊剥脱部位与临近剥脱部位内皮细胞阳性着色点无明显差异;实验组与对照组相比球囊剥脱部位内膜的阳性着色减少,内皮下层出现散在的阳性着色点,而临近球囊剥脱部位内膜阳性着色点较对照组无明显差异。说明高脂血症可以导致血浆NO水平下降,但对血管内皮eNOS酶表达无明显影响,只有在血管内皮损伤后,血浆胆固醇进入内膜进一步演变成有害胆固醇蛋白,进而对内皮功能产生影响。
2.兔动脉粥样硬化与炎症及细胞免疫的关系
通过ELISA方法检测实验组血浆炎症因子表达水平,并流式细胞检测技术和免疫组织化学方法,分析AS动物模型血管壁内DCs及免疫细胞的分布,观察是否存在与正常对照组之间的区别。
2.1血生化指标及炎症指标的检测:
制备模型2月后,通过ELISA方法检测实验动物血浆炎症因子表达水平,实验组兔血清sICAM、sVCAM、IL-6、TNF、hsCRP的含量与对照组相比,存在显著性差异(P＜0.01)。提示在兔动脉粥样硬化的发生、发展过程中存在炎症反应,黏附分子与CRP等炎症因子共同促进动脉粥样硬化的发展。
2.2兔DCs表型分子S100及抗原呈递细胞活性分子CD86、MHCⅡ分别在两组剥脱及未剥脱血管部位的表达情况
制备模型2月后,经流式细胞术检测,实验组血管剥脱部位的CD86、MHCⅡ、S100含量分别与未剥脱部位、对照组剥脱部位的血管相比明显升高,差异有显著意义(P＜0.01);CD86、MHCⅡ、S100的含量在实验组未剥脱部位、对照组剥脱部位及未剥脱部位的血管两两之间,差异无显著性意义(P＞0.05)。在模型兔剥脱部位血管壁组织内检测到兔特异性的DCs标记分子明显增多,说明实验组较对照兔腹主动脉血管壁内DCs明显升高;而检测抗原呈递细胞活性分子CD86及MHCⅡ含量也较对照组明显升高,说明抗原呈递细胞活化与AS的进展高度相关
3.兔外周血来源的DCs可以在模型自体血清及AS相关物质刺激下诱导成熟,并具有诱导T细胞增殖的能力
制模前兔外周血来源的DCs分为4组,空白组予以PBS干预,对照组予以制模前的兔自体血清干预(10%),实验组予以制模后兔自体血清干预(10%),ox-LDL组予以ox-LDL(10μg/mL)干预。
3.1兔外周血源的DCs培养及形态学鉴定:
兔外周血源的DCs在光学显微镜下可见悬浮生长,部分细胞聚集成簇,细胞表面有数量不等、形态不一的树突样突起;在扫描电镜下细胞呈不规则形,表面粗糙,胞体有形态不一的突起。
3.2各组兔外周血源的DCs的表型流式细胞学检测:
空白组、实验组、对照组以及ox-LDL组的兔外周血源的细胞培养前后的表型流式细胞学检测结果经One-Way ANOVA检验显示:MHCⅡ(P=0.000)、CD86(P=0.001)、CD14(P=0.000)在各自的不同组间的差异有显著性意义,对照组、实验组与ox-LDL组的MHCⅡ、CD86的表达较空白组上调,差异有显著性意义(P<0.05),实验组与ox-LDL组的CD14表达下调明显,差异有显著性意义(P<0.01)。对照组表现为CD86~+/MHCⅡ~+/CD14~(dim),区别于空白组单核/巨噬细胞的MHCⅡ~+/CD14~+的表型特征。在实验组自体血清环境以及ox-LDL组的ox-LDL刺激后,MHCⅡ和CD86表达继续上调,CD14~+/aim则转化为CD14~-,其MHCⅡ~+/CD86~+/CD14~-的表达为成熟树突状细胞的表现。
3.3混合淋巴细胞反应:
各组的兔细胞按照不同比例(1:10、1:50、1:100)与同种异源的T淋巴细胞混合,加DMSO后用酶标仪检测波长490nm的A值。结果显示各组均可产生不同程度的增殖反应,析因分析表明混合比例因素(P=0.000)和分组因素(P=0.000)对MLR的结果影响均有显著性意义,且1:10的比例时增殖反应最强。One-Way ANOVA检验各组在不同混合比例下的差异,实验组(P=0.026)各比例间差异有显著性意义。One-Way ANOVA检验不同比例的MLR在各组之间的差异,结果提示在1:10及1:50混合比例时各组之间比较有显著性意义(P=0.002),且ox-LDL组和实验组较空白组具有更强的刺激异体淋巴细胞增殖的能力,比较有显著性意义(P<0.05),说明经高脂血清环境培养的DCs与ox-LDL刺激生成的DCs具有更强的抗原提呈能力,并且随着DCs数量的增多,刺激同种异体淋巴细胞增殖的能力增强。
通过上述三个部分的实验,我们得出以下结论:
1通过球囊内皮剥脱并高脂饮食2月后,可形成明确的兔腹主动脉硬化斑块,该斑块具有显著的内膜增生、管腔狭窄等特点;
2模型兔腹主动脉斑块具有部分类似人动脉硬化的特点,体表超声显示该斑块呈密度低、偏心形等特点,麦角新碱诱发下腹主动脉造影显示球囊内皮剥脱部位可诱发明显的血管痉挛;
3斑块形成部位eNOS减少说明斑块部位内皮功能严重受损;
4模型兔血浆炎症因子分泌增多,说明炎症与AS形成高度相关;
5模型兔血管壁DCs的比例增多,且其活性表型分子表达增多提示DCs可能参与AS的形成;
6外周血分离的PBMC可在IL-4及GM-CSF的联合诱导下,培养出形态及表型稳定的DCs;
7模型兔自体血清可诱导DCs的成熟及促进T淋巴细胞的增殖,可能参与AS形成;
8 ox-LDL同样可诱导DCs的成熟及免疫活性,可能在本实验动物模型中占据主要的抗原成分地位。
Coronary heart disease(CHD)has been the greatest threaten to human beings since the arrival of the 21th century.However,the pathogenesis of CHD has not been clearly identified,so it is a great hindrance to the treatment of CHD.Thus,to study on the pathogenesis of CHD has become a pop trend of cardiology.It is generally accepted that atherosclerosis(AS)is the pathologic basis of cardiac cerebro vascular disease.AS is considered to be a progressing course which including the formation of fatty streak,fatty plaque,fibrous plaque,atherosclerotic plaque,and so on.People's initial recognition on AS is the anabrotic tumor-like proliferation of artery endomembrane in the final stage by clinical autopsy of dead cases.Subsequently, people discovered fatty streak and fatty plaque on arterial vessel walls in young cases which died of any disease but CHD.Finally,people got to know that AS proceeds slowly and gradually by making animal experiments and further clinical observations. Clinical observations in recent years had found that end stage CHD would have an accelerated progress which appears as sudden onset of an event with a high rate of risk,and so on.The mortality and mutilation rate will be surprising high if the salvages and treatments are not available in time.The predominant pathogenic characteristic of the arteries in these cases is the forming of unstable plaques.Plaques in this situation are always fragile so they may disrupt easily and quickly.Eventually, a chute-like thrombogenesis which will worsen the disease is caused.People again realized that end stage CHD may evolve acceleratedly and progress intensively.So we call this kind of end stage CHD acute coronary syndrome(ACS).The concept is emphasizing on a worsen stage in the developmental course of CHD.The clinical symptoms and signs are larvaceous in the initial and advancing stages of CHD,but when it comes to the end stage,the mortality and mutilation rate is getting surprisingly high,making great damage or loss to people' daily life or work.
AS experiences a slow and continuous pathological change,but may speed up in the end stage of its natural course.People found that pathological process of this kind has some similarity to that of trauma,tumor,inflammation,or autoimmune diseases, and so on.So different kinds of presumes on the morbility and progressing mechanisms of AS emerged,such as theories of trauma-like disease,tumor-like disease,inflammation-like disease,or autoimmune disease,just name a few.However, no even a literature documents that AS is a process of repair in any kind of trauma for that AS advances to a more serious stage when more repairs are done.No one confirms that AS is a kind of tumor for that more cells will experience apoptosis with more cellular proliferation.No one had even found out an antigen that would generally be accepted if it is proved to be a chronic inflammation or an autoimmune disease.Any of these theory faces a difficulty in explaining the morbility and advancing mechanism of AS.So how to make a breakthrough to find out a more convincing theory to help the explanation of AS pathogenesis is a difficulty to any one of the existed theory.Detections on the activated immunocells and cytokines in vivo in ACS cases and,detections made recently on the activated immunocytes in different stages of AS indicate that not only can we detected a great sum of activated T cell and monocytes,but also we can detected a high level expression of pro-inflammatory factors,in the peripheral blood,this suggests that a intensified inflammatory reaction in the body.With more knowledge on activated T cells are obtained,people pay more attention to the roles of inflammation and immunity in the development of AS than ever before.Studies on T cell subgroup,T cell typing, receptor expression and adhension factor had found that a great sum of activated T cells appeared in the peripheral blood and affected tissues of ACS cases.The difference in subgroups,typing and protein expression can also be noticed.The desire to know what activate T cells makes people pay more and more attention to the existence of dendritic cells(DCs).DC is a main kind of antigen presenting cell(APC) which are capable to induce the activation of T cells.Questions as whether DCs exist in the AS plaques or not,what is the origin of DCs,whethr DCs are in a mature status or not and what are their functioning status,whether DCs phagocytize the antigens and then present the antigen peptide-stimulated signs to incipient T cells so as to induce the proliferation and activation of T cells,have becoming the focus presently.
Normally speaking,DCs can hardly be seen in all kinds of body tissues.DCs are less than 1%of all the karyocytes in peripheral blood.It is even harder to find out any DCs in the normal vessel walls.All of these make it difficult to make a further study on DCs.Some scholars obtain DCs in vitro by cultivation of mononuclear cells from peripheral blood.They compared the maturity and immune functions of DCs in different stages of AS.They found that the PBMC derived DCs in ACS cases showed a high expression of costimulatory molecules and adhension molecules such as CD80, CD86,CD83,and so on.What's more,they had found that the DCs appeared to be mature and had a strong ability to activate T cells.All of these suggest that some substances which are capable to induce the activation of DCs and make them appear to be mature,exist in ACS cases.AS-related substances such as oxidated low density lipoprotein,nicotine and heat shock proteins,may induce the maturity of DCs and, give them the immune activity.However,if the study just retents on the in vitro experiments which may only confirm that the AS-induced antigen stimulus play roles in the activation of cells,or only makes a detection on the maturity standards of PBMC derived DCs cultured in vitro in ACS cases,no further progress will be made on the forming and advancing mechanism of AS.So,the questions mentioned above still can't be confirmed.
In order to further confirm that DCs certainly play a role in the progress of AS, and that they initiate and activate T cells in the forming of unstable plaques in ACS cases so as to intensify the inflammatory reaction and,on the basis that DCs may experience a developmental process of transforming from bone marrow stem cells to DCs precursor cells in vivo firstly,and then they will deadexis to the antigen-located sites,then the immature DCs may phagocytize and present the antigens so as to become mature cells which will deadexis to the lymph node T cell regions subsequently to activate incipient T cells for their further proliferation.We make a hypothesis that for verifing the distribution of DCs in peripheral blood and reflection its movement from side face into the vessel wall,and investigating the affection of the possible risk factors of AS for the maturation of the DCs and immune activation in vitro,we should analyse the relationship between typical plaque and inflammation in animal model which is suitable for researching the AS,and further to analyse local inflammation of plaque and the distribution and function of DCs.If DCs aggregate and be activated by phagocytizing antigens in the plaques in ACS cases, their distribution levels in vessel walls may raise,so a redistribution of DCs in peripheral blood and lymphs can be noticed.We started our study with the presume that detection of DCs proportion and quantity in different stages and different tissues in their genesis and developing courses would help to investigate the roles of DCs in the forming of unstable plaques in AS,so it may help to set up a experimental ground for the establishment of a confirmed immune mechanism of CHD.
Our experiments can be divided into three portions.First of all,to set up basic research model,we should take the rabbit model abdominal of aorta atherosclerosis as the objection,and apply the pathology slice,angiography and vitro ultrasound for detection the plaque formation and analysis the nature of the plaque;the distribution of DCs in the blood,vascular tissue,spleen and liver and lymph nodes and stressly the impact of the distribution of DCs on the generation and development of AS should be investigated by flow cytometry and immunohistochemistry from setting up the laboratory animals.For elucidating the possible mechanism of the formation of plaque,cuture the bone marrow-derived DCs in vitro,and observation the maturation of DCs stimulated by OxLDL through ELISA,flow cytometry and ELISA should be done.The results are as follows:
1 Preparation the New Zealand rabbit model and related testing
1.1 Established status of the New Zealand rabbits model of atherosclerosis and comparison the general situation in each group.
16 pure-bred New Zealand white male,weighing 2.50±0.14kg,are randomly divided into control group and experimental group.After injuring the intima of abdominal aorta of the rabbit by balloon,high-fat diet should be fed with for 2 months in experimental group,and normal diet should be fed with in control group.After giving high-cholesterol diet for 2 months,the body weight of rabbits after feeding than before both in the experi-mental group and the control group are significantly increased,and have significant difference(P=0.000);the levels of TC, TG,LDL in the experimental group after feeding higher than before respectively, and have significant difference(P=0.000);the levels of TC,TG,LDL in the experimental group are significantly increased than in the control group respectively, and have the significant difference(P=0.000).
1.2 Comparison the result of angiography in abdominal aortic and peripheral vascular ultrasound imaging of the New Zealand white rabbits in each group.New Zealand White rabbits are fed up for 2 months,then we take the angiography in abdominal aortic and peripheral vascular ultrasound for comparing the influence of atherslerosis plaque formation on abdominal aortic lumen and the ergometrine-induced spasm in site where denudated by balloon in two groups of New Zealand White rabbits.
The drug-stimulated vascular contraction rate is measured in abdominal aortic angiography,and there have significant difference between injuried sites and uninjuried sites of the experimental group(P=0.000),and injuried sites of the experimental group and the control group(P=0.000)by t tests.
Blood vessels eccentricity index is evaluated by ultrasound in the body surface (plaque eccentricity index=plaque thickness of the thickest plaque site/plaque thickness of the contralateral site of thinnest site).The results show that a larger plaque eccentricity index have a marked increase in vasoconstriction that stimulated by drug,and eccentricity index rate positive correlated significantly with vasoconstriction(r=0.983,P=0.000).
1.3 Comparision the intimal hyperplasia of abdominal aortic of New Zealand white rabbits in each group
The specimens of the near site and the site of balloon denudation of the abdominal aortic endarterectomy should be collected in two groups after feeding up the New Zealand white rabbits for 2 months,respectively.Then the aortic morphology shoud be compared,membrane and intimal thickener significantly in experimental group in the light microscope,vascular smooth muscle cells proliferated more,and arranged neatly in ring,a large number of extracellular matrix formed in neointimal,and narrowed the lumen significantly.
There have significant difference in parameters such as around area of the internal elastic membrane,neointimal area,lumen area and the percentage of intimal hyperplasia area to lumen area among four groups,the neointimal area increased significantly higher in the site and the near site of endarterectomy by balloon in exprimental group than in the control group,and the difference is significant(P= 0.004)in independent samples t test,as well as the percentage of the intima hyperplasia(P=0.001),but lumen area decreased(P=0.000),indicating that neointimal is generated in the aorta denudation site by balloon in experimental New Zealand rabbit group.
1.4 Comparison the levels of NO in serum and eNOS protein expression in vascular specimens of New Zealand white rabbits in each group.
After feeding up New Zealand white rabbits for 2 month,the levels of serum NO in 2 group detected by nitrate reductase method.And the results showed that compared with the control group,the levels of serum NO in the experimental group have significant dicreased(P=0.002).The eNOS protein in vascular tissue are detected in quantity and location by immunohistochemistry for each group,and the results show that,endothelial cells have stained positively in vascular tissue of the control group,and the positive stained sites of endothelial cells have no significant difference in and near in balloon endarterectomy site.Compared with the control group,positive staining point of the intima in balloon endarterectomy site have reduced in the experimental goup,and the positive staining points scatter in the lower endothelial,but in the near site of balloon endarterectomy have no significant difference.It indicated that hyperlipidemia could lead to a reduction in the levels of serum NO,but have no significant effect on the expression of vascular endothelial eNOS enzyme,only in the vascular endothelial injuried,cholesterol could migrate from the serum into the intima and further evolute into harmful cholesterol protein, and thus have an influence on endothelial function.
2 The relationship between rabbit atherosclerosis and inflammation,cell immunity The existence difference between the normal control group and the experimental group are observed though detection the expression levels of inflammatory cytokines in serum by ELISA,and analysis the distribution of DCs and immune cells in the AS vessel wall by flow cytometry and immunohistochemistry.
2.1 Detection the index of blood biochemical and inflammation The levels of serum inflammatory cytokines are detected by ELISA after preparation the model for 2 months,and there have significant difference in the content of serum sICAM,sVCAM,IL-6,TNF,HsCRP between experimental group and control group(P＜0.01).It indicated that inflammatory response exist in generate and development of AS,and adhesion molecules,CRP and other inflammatory cytokines promote the development of AS.
2.2 The expression of S100 of DCs phenotype molecular and CD86,MHCII of antigen presenting moleculars in and near in vascular endarterectomy site
Detection the expression levels of MHCⅡ,CD86,S100 by flow cytometry after preparation model for 2 months,and the levels of MHCⅡ,CD86,S100 are higher in endarterectomy site in experimental group than near in endarterectomy site in experimental group,endarterectomy site in control group(P＜0.0l),but have no significant difference among near in endarterectomy site in experimental group, endarterectomy site in control group,near in endarterectomy site in control group(P＞0.05).The rabbit-specific molecular markers of DCs markedly increased in endarterectomy site in the rabbit model of vessel wall tissue,indicating that the expression of DCs are increased more in abdominal aorta vascular wall than control group.The content of CD86 and MHCII molecules of antigen-presenting cell activity moleculars are also significantly higher than the control group,indicating that activation of antigen-presenting cells is highly relevant to the development of AS.
3 The peripheral blood DCs of rabbit can be stimulated and induced to maturation by AS related substance,and have the ability to induce T cell proliferation
3.1 Culture and identify the rabbits peripheral blood derived DCs
The cultured rabbit peripheral blood cells is that suspended growth,some clustered together and different quantity and shape in dendritic-like protrusion on the surface,which can be seen under the light microscope,and irregular in shape,rough surface,different patterns of protrusion under scanning electron microscopy.
3.2 Detection the phenotype of rabbits peripheral blood derived DCs by flow cytometry in each group
The phenotype of rabbit peripheral blood cell which before and after cuture in the experimental group,the control group and the LPS group are detected by flow cytometry,and the results are tested by One-Way ANOVA and showed that:MHCⅡ(P=0.000),CD86(P=0.001),CD14(P=0.000)have significant difference between groups,respectively;compared with control group,the expression of MHCII,CD86 are upregulated in the experimental group and the ox-LDL group,and the difference is significant(P＜0.05),the expression of CD14 are significantly reduced in experimental group and ox-LDL group,and the difference is significant (P＜0.01).The phenotype of the control group is CD86~+/MHCⅡ~+/CD14~(dim),and different from monocytes/macrophages cells with MHCⅡ~+/CD14~+phenotype in the control group.Experimental group at the environment and the autologous serum ox-LDL group of ox-LDL stimulation,The expression MHCⅡand CD86 remain increased,and CD14~+/~(dim)is tranformed into CD14~-,and the phenotype of MHCⅡ~+/ CD86~+/CD14~-is the matured dendritic cells expression.
3.3 Mixed allogenic Lymphocyte reaction
According to the different proportion(l:10,1:50,1:100),each group rabbit cells are mixed with allogenic T-lymphocytes,A value is detected at 490nm wavelength by microplate reader after adding DMSO.The results show that proliferative response is vary in each group,showed that the proportion of factors(P=0.000)and group factors(P=0.000)have impact on the results of MLR by factorial analysis, and the significance is significant,and at the ratio of 1:10,the proliferative response is the strongest.There have significant difference among the different mixed ratio in the experimental group(P=0.026)by One-Way ANOVA test.Compared the defference of MLR in each group by One-Way ANOVA test,and the results suggest that at the mixed ratio of 1:10 and 1:50,the difference is significant among groups(P=0.002),and the ability to stimulate allogeneic lymphocyte to proliferate is more stronger in the ox-LDL group and the experimental group than in the control group,and the difference is significant(P＜0.05),suggesting that DCs cultured with high fat environment and stimulated by ox-LDL have more ability to present antigen, and with the increase of DCs,the ability to stimulate allogeneic lymphocyte proliferation is strengthen.
Through above three-part test,we draw the following conclusions:
1 Through endothelial denudation by balloon and high-fat diet for 2 months, rabbit abdominal atheroslerosis plaque may be the generation,and the plaque are characterized by significant intimal hyperplasia and narrow lumen;
2 The characteristics of rabbit model of abdominal aortic arteriosclerosis plaque are partly similar with people,the plaque density was low and with eccentric shape which showed by surface ultrasound,and with induced by ergonovine,the balloon-induced endothelial denudation site showed significantly of vasospasm by abdominal aortic angiography;
3 The site of plaque generation have lower eNOS,indicating that endothelial function are damaged severely;
4 The secretion of inflammatory cytokines are increased in the plasma of rabbit mode,suggesting that inflammation high associated with the generation of AS;
5 The proportion of DCs are increased in the vessel wall of rabbit model of and the molecular phenotype of DCs are activated,suggesting that DCs may be involved in the generation of AS;
6 Peripheral blood derived PBMC can be induced by IL-4 and GM-CSF into a stable morphology and phenotype of DCs;
7 Autologous serum of rabbit model can induce maturation of DCs and promote the proliferation of T lymphocytes,thereby may be involved in the generation of AS;
8 ox-LDL can induce maturation and immune activity of DCs,and probably occupy the status of the main components of the antigen in this experimental animal models.
引文
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