摘要
背景
心力衰竭(简称心衰)是扩张型心肌病(DCM)、冠心病、风湿性心脏病、高血压心脏病等多种心血管疾病发展的共同终末阶段,对人类生命构成巨大威胁,已成为全球范围内造成死亡的主要原因之一。心衰的发病过程非常复杂,已知的心衰发生机制主要有心肌损害、血液动力学异常、神经激素的激活和心肌重构等。一项长达10年的临床研究表明,尽管针对心衰发病的上述机制采取了许多积极的治疗措施,但心衰病人的预后并没有明显的改善,其存活率与10年前基本相同。这一结果说明,真正降低心衰的患病率和死亡率非常困难,提示在心衰发生发展过程中还有许多未知因素的存在。进一步探讨这些未知因素,对从根本上预防和治疗心衰将具有重要的理论与实践意义。
β肾上腺素受体(βAR)是交感神经系统的重要成员,通过介导体内儿茶酚胺类物质的生理效应,在调节心脏活动中发挥着重要的作用。βAR属于G蛋白偶联受体家族,分为β_1、β_2和β_3AR三个亚型,具有相同的结构特征,即含有7个22～28个氨基酸的跨膜区、3个细胞内环和3个细胞外环。以前认为在心脏中βAR为β_1AR和β_2AR两种亚型,通过兴奋型G蛋白—腺苷酸环化酶—环磷酸腺苷系统,发挥心肌正性变时、变力和变传导效应。最近的研究发现,在人类等多种种属动物的心肌细胞中有β_3AR的表达,但与经典的β_1AR和β_2AR不同的是,人类心室肌组织β_3AR通过与抑制型G蛋白(inhibitory G protcins,Gi)偶联,活化eNOS-NO-cGMP通路,介导心肌负性变力效应。此外,由于β_3AR是一种低亲和力受体,即激活β_3AR所需儿茶酚胺的浓度明显高于激活β_1AR和β_2AR所需的浓度,因此在生理情况下,β_3AR调节心脏功能的意义可能不大。
在心衰发生发展过程中,交感神经系统活性过度增高,心脏β_1AR和β_2AR由于PKA和βARK的磷酸化等机制而脱敏,受体密度下调,使这些受体对儿茶酚胺的反应性减弱,从而促使机体产生更高浓度的儿茶酚胺类物质。与此相反的是,心衰时β_3AR表达水平较正常人上调2—3倍,而且与之偶联的G_1蛋白也上调,再加上此时也具备激活β_3AR的配体条件—高浓度的儿茶酚胺,所以在心衰时β_3AR很有可能被激活。在人类衰竭的左心室中,β_1-和β_3AR相反的改变导致他们的变力效应不平衡,有可能成为加重心脏功能恶化的因素。但在β_3AR转基因小鼠模型中,并没有检测到心肌细胞肥大增生、纤维化等组织学改变,激活β_3AR可能不是引发心脏损伤的原因。这提示在心衰发生发展过程的不同阶段,β_3AR的活化可能发挥着不同的病理生理作用。
上世纪90年代以来,国内外学者相继发现在DCM等心脏疾病患者血清中存在有β肾上腺素受体(β_1和β_2AR)的自身抗体,这些自身抗体可通过作用于相应受体细胞外第二环而产生类激动剂样效应,且采用相应细胞外第二环肽段长期免疫动物,可使其心脏功能及形态均发生类似DCM病人的变化。由于G蛋白偶联受体家族成员具有相似的结构特征,为证实心衰发病过程中是否有针对β_3AR的自身免疫作用的参与,2005年我们以人工合成的人β_3AR细胞外第二环的肽段作为抗原,采用酶联免疫吸附实验(ELISA)筛查了217例心衰病人和100例正常人血清中的抗β_3AR抗体。结果发现,心衰患者血清中抗β_3AR抗体的阳性率明显高于正常人(27%vs11%,P＜0.05)。我们的研究还发现,该抗体具有类β_3AR激动剂的负性变时、变力效应,且此效应具有不脱敏现象。但是,上述实验结果还不足以说明针对β_3AR细胞外第二环的自身抗体与心衰发病的关系。
首先,由于心衰的发病原因不同,其机制及病理改变也存在一定差异。高血压、冠心病、扩心病、肥厚性心肌病及风心病等多种疾病所致心衰的发病过程中均存在免疫系统紊乱,并已得到该研究领域国内外专家的公认,但针对心肌的自身免疫作用的研究结果却不尽相同。例如Rahat S等的研究结果显示抗肌球蛋白抗体在冠心病与扩张性心肌病所致心衰患者中的阳性率及亲和力无明显差异;而Jonathan H等则报道扩张性心肌病所致心衰患者中抗肌球蛋白抗体的阳性率及亲和力高于冠心病所致心衰患者。另Jahns R和Fu ML等的研究结果则显示:虽然继发于扩心病、冠心病、高血压和肥厚性心肌病等疾病的心衰患者体内均检测到β_1AR自身抗体,但此抗体却只在扩心病及冠心病心衰过程中发挥作用,而与继发于高血压和肥厚性心肌病的心衰发病无关。那么,在不同原因所致心衰过程中,β_3AR自身抗体的阳性率及亲和力是否有所差异,β_3AR自身抗体在何种疾病所致心衰的发生发展过程中对心脏功能产生影响尚待进一步证实。
其次,尽管诸多研究表明,G蛋白耦联受体细胞外第二环具有T细胞及B细胞表位,有很强的抗原性和免疫原性,是产生自身抗体的主要表位。但也有研究证实,DCM患者体内同样存在针对β_1AR细胞外第一环的功能性自身抗体。考虑到β_3AR细胞外三个环均具有暴露于机体免疫系统的可能,那么,它们是否均能刺激机体产生相应自身抗体?如果存在这些自身抗体,其各自有何临床意义值得我们去关注。目前关于针对β_3AR细胞外第一、第二和第三环自身抗体产生情况的比较研究尚未见有报道。
再次,自发现β_1AR自身抗体至今已经历了19年的时间,期间又发现了诸多针对G蛋白偶联受体细胞外第二环的自身抗体,并对它们相应的临床意义和病理生理机制进行了大量的研究,但到目前为止,自身抗体产生的免疫学机制还不十分明确。抗原的数量、位置及机体免疫系统功能的变化均是调节抗体产生的重要因素。在正常机体内存在针对自身组织的自身反应性淋巴细胞,当自身抗原存在量达较高危险浓度时就有可能活化相应的自身反应性淋巴细胞,产生自身抗体。而B淋巴细胞是机体体液免疫应答过程中产生抗体的主体,其数量或/和抗体产生能力的改变都将导致机体抗体水平的改变。业已证实,在心衰发生发展过程中,心肌细胞β_3AR mRNA和蛋白质水平均有增高,同时机体免疫系统存在持续激活,这些条件可能会导致机体针对β_3AR的免疫反应性增加,进而产生针对β_3AR的自身抗体,但这一假说需要进一步的研究证实。
此外,虽然我们的前期研究证实,β_3AR自身抗体具有受体激动剂样效应,可以影响离体心肌细胞活动产生类激动剂样的生物效应,但在整体动物,β_3AR自身抗体长期存在下,会对心脏结构及功能产生什么样的作用,尚不清楚。而对这一问题的解释,有利于全面评价β_3AR自身抗体存在的可能的病理生理意义,以及对β_3AR自身抗体进行深入研究提供方向。
综上所述,我们拟在在本课题进行以下三方面的研究:①不同原发疾病所致心衰患者血清中,β_3AR自身抗体分布的免疫学特征及其与心衰某些特异性指征的相关性分析,借以分析β_3AR自身抗体在不同原发疾病心衰发生中的可能作用;②在β_3AR自身抗体阴性的大鼠,建立增大心脏压力后负荷致心衰的动物模型,观察在心衰的发生过程中是否会产生β_3AR的自身抗体,如果产生,其产生规律为何;③通过用β_3AR细胞外环肽段主动免疫正常大鼠,观察在β_3AR自身抗体长期存在下,心脏结构和功能是否会发生改变。
一、心力衰竭患者血清中β_3AR自身抗体的种类和可能的临床意义
目的
本研究分别以合成的人β_3AR细胞外第一环、第二环和第三环表位肽段作为抗原,应用酶联免疫吸附测定(ELISA)技术,检测不同原发疾病所致心衰患者血清中β_3AR的自身抗体,并分析抗β_3AR自身抗体的免疫反应性及其对心脏功能活动的影响,以探讨心衰患者血清中抗β_3AR自身抗体存在的病理生理意义。
方法
1.研究对象及标本
166例继发于扩张型心肌病(简称扩心病,n=30)、冠状动脉粥样硬化性心脏病(简称冠心病,n=93)、高血压性心脏病(简称高心病,n=33)和风湿性心脏病(简称风心病,n=10)的心衰患者为山西医科大学第一医院、山西省人民医院及山西省心血管疾病研究所2005～2007年的住院病人,心功能Ⅱ～Ⅳ级,左室射血分数(LVEF)均低于45%(病例资料见表1)。全部患者除外内分泌、自身免疫性疾病、各种感染性疾病和免疫缺陷性疾病,采集血样前3d避免服用免疫抑制剂和βAR阻滞剂。正常对照组:健康体检者103例,年龄、性别构成与心衰组均衡,无任何临床症状且心电图、超声心动图、X线胸片、肝肾功能常规检查未见异常。入选者均于空腹12h后采集静脉血液并及时分离血清置于-20℃保存。
2.肽段合成
3个抗原肽段均由西安美联生物工程公司合成,相当于人β_3AR细胞外第一环、第二环及第三环氨基酸序列的特异性抗原决定簇,合成肽段纯度为95%。人β_3AR氨基酸序列检索来自NCBI(AC:P13945,GI:461604)(见表2)。合成的肽段储存于-20℃备用。
3.检测方法
按照我实验室以前的方法,应用SA-ELISA检测方法。抗原包被用量为5μg/ml。主要技术流程如下:包被合成的抗原多肽→加入按一定比例稀释的待测血清→加入标记有生物素的抗人IgG抗体→加入标记有辣根过氧化物酶的亲和素→加入辣根过氧化物酶的底物及显色剂→在酶标仪上测定反应物的光密度值(optical density,OD)。
4.判断标准
抗体水平以标本1:20稀释时测定的OD值反映。以阳性血清与阴性血清的吸光度之比即P/N比值来判断阳性率,P/N≥2.1为阳性(P/N=标本OD值-空白对照OD值/阴性对照OD值-空白对照OD值)。
5.统计学处理
采用SPSS 13.0统计软件对实验数据进行统计。率的比较采用x~2检验或Fisher's确切概率法;计数资料均数间显著性差异比较采用方差分析或t检验,等级资料采用秩和检验。
结果
1.正常人和心衰患者血清中β_3AR自身抗体的种类及阳性率
经ELISA筛选发现,心衰患者及正常人血清中均存在分别针对β_3AR细胞外第一环、第二环和第三环的自身抗体(依次表示为anti-β_3-EC_Ⅰ,anti-β_3-EC_Ⅱ和anti-β_3-EC_Ⅲ),但各自阳性率不同。其中,扩心病患者anti-β_3-EC_Ⅰ及anti-β_3-EC_Ⅱ阳性率(33.3%和40.0%)均高于正常人(分别为9.7%和8.7%,P＜0.05),而anti-β_3-EC_Ⅲ阳性率(6.7%)与正常人(5.8%)无差异(P＞0.05);冠心病患者anti-β_3-EC_Ⅱ的阳性率(28.0%)也显著高于正常人(8.7%,P＜0.05),而anti-β_3-EC_Ⅰ及anti-β_3-EC_Ⅱ阳性率(19.4%和3.2%)和正常人相比无明显差异(P＞0.05);高心病和风心病患者血清中anti-β_3-EC_Ⅰ,anti-β_3-EC_Ⅱ和anti-β_3-EC_Ⅲ的阳性率与正常对照比较经x~2检验(或Fisher's确切概率法)均无差异(P＞0.05)(见图1)。
这一结果提示β_3AR自身抗体(主要为anti-β_3-EC_Ⅱ抗体)可能与扩心病及冠心病所致心衰的发病过程有关。
2.正常人和心衰患者β_3AR自身抗体的水平
分析β_3AR自身抗体阳性血清中的β_3AR自身抗体水平(见图2)发现:正常人组内anti-β_3-EC_Ⅱ的抗体水平(0.92±0.23)均明显高于anti-β_3-EC_Ⅰ(0.28±0.06)和anti-β_3-EC_Ⅲ(0.35±0.05,P＜0.05),同样在各心衰患者组内anti-β_3-EC_Ⅱ的抗体水平也均明显高于anti-β_3-EC_Ⅰ和anti-β_3-EC_Ⅲ。提示正常状态下β_3AR细胞外第二环就具有较强的免疫性,anti-β_3-EC_Ⅱ具有较强的亲和力,更易于和相应抗原结合而被检测出。此外,在扩心病患者血清中的anti-β_3-EC_Ⅱ水平(1.17±0.17)显著高于正常人(0.92±0.23,P＜0.05),其他三种疾病所致心衰患者的anti-β_3-EC_Ⅱ抗体水平和正常人无明显差异(P＞0.05);而anti-β_3-EC_Ⅰ和anti-β_3-EC_Ⅲ的抗体水平在正常人和各种原发疾病所致心衰患者间均无明显差异(P＞0.05)。
上述结果均提示,anti-β_3-EC_Ⅱ是与心衰(尤其是扩心病所致心衰)发病过程有关的主要的β_3AR自身抗体的种类。
3.β_3AR自身抗体与心衰患者心功能之间的关系
以心脏左室射血分数(ejection fraction,EF)和NYHA(纽约心脏协会)心功能分级作为反映患者心功能状态的指标,分析在不同原发疾病所致心衰中各类β_3AN自身抗体(anti-β_3-EC_Ⅰ,anti-β_3-EC_Ⅱ和anti-β_3-EC_Ⅲ)对心功能的影响。经t检验及秩和检验发现,在扩心病患者中,anti-β_3-EC_Ⅱ阳性者较阴性者心功能好,表现为anti-β_3-EC_Ⅱ阳性者EF显著高于阴性者(P＜0.05),同时NYHA心功能分级低于阴性者(P＜0.05);然而,anti-β_3-EC_Ⅰ阳性者较阴性者心功能差,表现为EF低于阴性者(P＜0.05),而NYHA心功能分级则高于阴性者(P＜0.05);冠心病患者中,anti-β_3-EC_Ⅱ阳性者也优于阴性者,但不具有统计学意义(P＞0.05),而anti-β_3-EC_Ⅰ阳性者心功能则低于阴性者(P＜0.05)(见表3);风心病及高心病患者β_3AR自身抗体阳性患者和阴性患者间心功能指标无差别。
这一结果提示,β_3AR自身抗体(主要是anti-β_3-EC_Ⅱ)与扩心病及冠心病所致的心衰有关,心衰发病过程中,anti-β_3-EC_Ⅱ可能对由β_1AR自身抗体诱发的心脏功能障碍和心肌损伤中起着心肌保护的作用。关于anti-β_3-EC_Ⅰ对心功能的影响,由于其抗体水平较低,观察例数较少,且其功能效应还不明确,故尚难得出明确结论。
小结:
1.针对β_3AR细胞外第二环的自身抗体,在扩心病和冠心病所致心衰患者中的阳性率和/或抗体水平均明显高于正常人,提示它可能与心衰的发病过程有关。
2.针对β_3AR细胞外第一环的自身抗体,在扩心病患者中也有较高的阳性率,但抗体水平较低;针对β_3AR细胞外第三环的自身抗体则与心衰发病无关。
3.心衰发病过程中,anti-β_3-EC_Ⅱ可能对由β_1AR自身抗体诱发的心脏功能障碍和心肌损伤起着一定的心肌保护作用。
二、心衰的发生发展过程与β_3AR自身抗体产生的关系
目的
为进一步探讨β_3AR自身抗体与心衰发病的关系,我们还在β_3AR自身抗体阴性的大鼠身上制备了增大心脏压力后负荷的心衰模型,借以观察在心衰发生发展过程中,β_3AR自身抗体的产生规律,并分析β_3AR自身抗体的产生与心肌β_3AR密度及免疫系统机能变化的联系,以便查明β_3AR自身抗体在心衰发生发展过程中所起的作用,加深对心脏疾病发生过程中的自身免疫机制的理解,并为相关疾病的防治提供新思路。
方法
1.采用缩窄腹主动脉法制备心衰大鼠模型:
采用与第一部分相同的ELISA方法和判断标准确定为β_3AR自身抗体阴性的健康Wistar大鼠(雄性,10周龄,体重200-250g,山西医科大学动物中心提供)随机分为2组:(1)主动脉缩窄组(AB组,n=80):根据Doering等的方法实施腹主动脉环扎术。(2)伪手术对照组(sham组,n=40):仅分离腹主动脉而不结扎,其他操作与主动脉缩窄组相同。
2.大鼠血清中p3AR自身抗体的测定:
所有实验动物于手术前及手术后每两周采用剪尾法收集血清,-20℃备用,采用ELISA方法(与第一部分相同)检测模型形成前后针对β_3AR细胞外第二环的自身抗体(根据第一部分研究结果选择)。抗原肽段序列由西安美联生物工程公司合成,相当于大鼠β_3AR细胞外第二环氨基酸序列(176aa～200aa,RVGADAEAQECHSNPRCCSFASNMP,检索自NCBI AC:P26255,GI:543882),合成肽段纯度为95%。合成的肽段储存于-20℃备用。
3.大鼠在体心功能测定:
两组大鼠均于术后4周、8周、12周和16周称体重,用25%乌拉坦麻醉后,仰卧固定于手术台上,颈前正中作一约4厘米的切口,分离颈前肌群至气管,分离右侧颈总动脉并结扎远心端,近心端用小动脉夹阻断血流,用眼科剪剪V字形口,将充满肝素生理盐水的导管经右颈总动脉插入心室并结扎固定,打开动脉夹。导管的另一端借三通管连接P—50压力换能器,压力信号输入MS2000生物信号记录分析系统,记录各心功能参数,包括左心室收缩压(LVSP),左心室舒张末期压(LVEDP)以及室内压上升和下降的最大速率(±dp/dt_(max))。测定结束后,开胸,迅速取出心脏,洗净心腔内积血、擦干,去除心脏周围组织及血管,称量全心重,计算心脏重量与体重的比值,部分心肌组织固定于10%中性福尔马林,其余组织于-70℃保存备用。同时按无菌操作法分离大鼠脾脏。
4.心脏组织形态学检测:
大鼠心肌置于10%中性福尔马林固定48小时,常规脱水,石蜡包埋。制作4μm组织切片,采用Masson染色及HE染色,于光学显微镜下观察心肌形态,并利用图象分析技术进行心肌染色切片胶原容积百分比(CVF)的测定。
5.蛋白免疫印迹技术(Western Blot)测定心肌细胞β_3AR含量:
大鼠左心室组织细胞膜蛋白提取按照膜相/水相蛋白提取试剂盒(TansMem proteinisolation system instructions,Applygen Technologies Inc)说明书操作。蛋白含量采用BCA法测定。30μg变性蛋白经10%SDS-PAGE凝胶分离后转膜,分别与大鼠β_3AR特异性抗体及二抗孵育,采用化学发光法测定。
6.大鼠B淋巴细胞含量测定:
脾脏研磨成悬液,采用Ficol淋巴细胞分离液2500rpm离心30分钟,收获单个核细胞层细胞,洗涤两次,调整细胞数为1×10~7/ml。台盼兰染色查活细胞数应大于90%。取100μl脾淋巴细胞(1×10~6个)与适量PE标记的抗大鼠CD45R单克隆抗体混合,室温下避光孵育20分钟后洗涤,应用流式细胞仪FACSCalibur进行检测。Ceil quest收集细胞(总数10000个细胞),并分析流式结果。
7.B淋巴细胞抗体生成能力测定:
用20%绵羊红细胞(SRBC)生理盐水悬液腹腔免疫大鼠,4天后无菌取出脾脏,用RPMI1640洗一次后置平皿中不锈钢筛网上,用无菌注射器针芯研磨成悬液,RPMI1640洗涤两次,调整细胞数为5×10~6/ml。台盼兰染色查活细胞数应大于90%。按照Jerne和Nordin等改良的直接及间接溶血空斑实验测定B淋巴细胞抗体生成能力。空斑数量采用VilBer Lourmat capt软件测定.
结果
1.模型大鼠术后生存率:
实施腹主动脉狭窄术后第2周,手术组大鼠存活44只,伪手术组大鼠存活36只,存活率分别为55%(44/80)和90%(36/40)。之后,两组大鼠在处死前均未出现意外死亡。
2.大鼠心衰模型成功指标:
2.1大鼠心重/体重比:实施腹主动脉狭窄术后4周,手术组大鼠心脏重量与体重的比值(0.32±0.01)较同期伪手术组(0.27±0.01)显著增加(P＜0.05)。术后8周,手术组心脏重量的增加(0.36±0.01)与术后4周相比进一步加剧(P＜0.05);在术后12周和16周,手术组心脏重量与体重的比值(分别为0.34±0.01和0.34±0.02)与术后8周相比无统计学差异(P＞0.05),与同期假手术组相比(分别为0.26±0.01和0.27±0.01)仍有显著性差异(P＜0.01)(见图3)。
2.2心功能变化:实施腹主动脉狭窄术后4周,手术组大鼠的LVSP和±dp/dtmax均低于伪手术组,LVEDP高于伪手术组,但无统计学差异(P＞0.05);术后8周,手术组上述左室收缩、舒张功能各参数改变进一步加剧,与同期伪手术组具有明显差异;术后12周和16周,两组模型中反映左心室功能的上述各项指标与术后8周相比,心功能障碍无进一步加重的趋势,而与同期假手术组相比,各参数仍有明显的差异(P＜0.01)。见表4。
2.3心肌形态学改变:通过Masson染色和HE染色技术结合光学显微镜观察及图象分析技术等证实,腹主动脉缩窄手术组大鼠随着时间的发展,发生了明显的心肌细胞形态改变及胶原纤维化,同时伴随程度不同的淋巴细胞浸润(见表5,图4,图5)。
以上结果显示,心衰模型建立成功。
3.心衰模型形成过程中,大鼠血清中β_3AR自身抗体的产生和变化规律:
心衰模型大鼠血清中β_3AR自身抗体水平(O.D.值)在术后2周(0.37±0.01)开始与术前(0.20±0.01)及同期伪手术组(0.20±0.02)相比,即有明显升高(P＜0.05),之后平稳上升至8周时达峰值(0.71±0.04),并保持至10周(0.70±0.05)。术后12周,心衰模型手术组大鼠血清中β_3AR自身抗体开始下降(0.60±0.07),但直至16周(0.52±0.06)仍维持在高于术前水平,而在14周及16周与同期伪手术组相比已无统计学差异(P＞0.05)。伪手术组大鼠血清β_3AR自身抗体测定结果也呈现一种平稳升高的态势,但直至实验结束,其差别也未具有统计学意义(P＞0.05)(见图6)。
这一结果提示,在心衰发生发展过程中β_3AR自身抗体具有逐渐产生、缓慢增高、维持、自然消退的变化规律。
4.心衰模型形成过程中,心肌细胞β_3AR的含量改变:
与同期伪手术组大鼠相比,手术组大鼠心肌细胞膜β_3AR的含量在术后4周已有轻微增加;在术后8周明显升高,差异具有统计学意义(P＜0.05);术后12周和16周,β_3AR的含量一直持续在较高的水平(见图7)。而伪手术组心肌细胞膜β_3AR的含量在整个实验进程中均未发生明显改变(P＞0.05)。此外,β_3AR含量的上调高峰出现在术后8周,与β_3AR自身抗体到达高峰的时程相符,
这一结果提示,在心衰的发生发展过程中,β_3AR的上调和β_3AR自身抗体的生成之间,在时间点上有较明确的对应关系,说明二者在功能作用上的一致性。但由于实验设计中观察时间点的限制,本实验还无法分析β_3AR上调和β_3AR自身抗体产生之间是否有因果关系。
5.B淋巴细胞数量及抗体产生能力的改变:
采用流式细胞仪技术测定实验大鼠脾淋巴细胞中B细胞的数量,结果显示在整个实验过程中手术组大鼠与同期伪手术组相比,其脾脏B淋巴数目并未明显改变(P＞0.05)(见图8)。同时采用直接及间接溶血空斑实验来测定实验大鼠脾淋巴细胞的功能(即抗体生成能力),结果表明,与伪手术组比较,手术组大鼠直接溶血空斑数量在术后4周已有明显升高,术后8周达到最高峰,在术后12周降至与伪手术组相似的水平(图9);而手术组大鼠间接溶血空斑数目同样在术后4周明显升高,术后8周达到高峰,并保持至术后12周,在术后16周降至与伪手术组相似的水平(图10)。说明在实验大鼠心功能改变过程中出现免疫系统抗体生成能力的变化,而且抗体生成能力的变化与血清中β_3AR自身抗体的变化规律十分相似。
这一结果提示,β_3AR自身抗体的产生可能与心衰发生发展过程中机体免疫系统功能的改变具有内在联系。
小结
1.在建立压力后负荷型大鼠心衰模型的过程中,原为β_3AR自身抗体阴性的大鼠血清中开始出现高水平的β_3AR自身抗体,但该抗体具有一定的产生、维持和减退的自我消长的时间过程;
2.β_3AR的产生过程与脾淋巴细胞抗体生成能力的改变趋势一致,但与B淋巴细胞数量无关;
3.β_3AR自身抗体水平出现峰值的时间与大鼠心肌细胞膜受体β_3AR的上调时间一致,说明二者在功能作用上有一致性;
4.本实验证实在心衰的发生发展过程中,心脏的病理生理改变促进了β_3AR自身抗体的产生,但其启动β_3AR自身抗体产生的确切机制还有待进一步探讨。
三、β_3AR自身抗体长期存在对在体大鼠心脏结构及功能的影响
目的
我们的前期研究证实,心衰病人血清中存在β_3AR自身抗体,主要针对β_3AR细胞外第二环,具有类激动剂样负性变时、负性变力效应,且此效应具有不脱敏现象,与DCM及冠心病等患者的心衰发生发展过程可能有一定的关系。但在在体情况下,此抗体的长期持续作用会对心脏结构及功能产生何种影响尚不清楚,而此研究对进一步理解在心衰发展过程中不同类别的受体抗体是如何发挥相互作用的,进而加深对扩心病等心脏疾患发病中自身免疫机制的理解,可能具有较重要意义。因此,本研究采用β_3AR细胞外第二环的合成肽段主动免疫正常大鼠六个月,观察被免疫大鼠在β_3AR自身抗体的持续刺激下心脏结构及功能的改变,并探讨β_3AR自身抗体可能的信号转导通路,借以分析β_3AR自身抗体在心衰发病过程中可能的作用机制。
方法
1.动物免疫:
选用8周龄、重量为100-160g、健康雄性Wistar大鼠20只(山西医科大学动物中心提供),随机分成两组:对照组(n=8)和β_3AR免疫组(n=12)。β_3AR免疫组根据人β_3AR细胞外第二环第179～203位氨基酸残基的序列人工合成抗原进行免疫,首次免疫时用合成的抗原肽段(0.4μg/kg)加完全佐剂进行,2周后采用合成的抗原肽段加不完全佐剂进行加强免疫,以后每隔两周加强一次,共免疫6月(24周);对照组除不加抗原外,余同免疫组。
2.血清中β_3AR自身抗体的测定:
上述两组大鼠均于免疫前及每次加强免疫前1天鼠尾采血,分离血清,用SA-ELISA方法测定其血清β_3AR自身抗体水平,测定方法见第一部分方法3。
3.大鼠在体心功能测定:
分别于初次免疫后3月(12周)及6月(24周)两个时间点,随机选取实验动物进行在体心功能测定,具体操作同第二部分方法3。心功能测定结束处死大鼠,留取新鲜心肌组织,部分以适当方法固定用于光学显微镜检查和电子显微镜检查,其余组织置于-70℃保存备用。
4.心肌形态学指标的测定:
采用光学显微镜及电子显微镜技术测定。
5.心肌组织β_3AR及eNOS含量的测定:
采用WestemBlot技术进行检测。
6.心肌组织cAMP及cGMP含量测定:
采用放射免疫技术测定,操作过程严格按照试剂盒说明书进行。
7.心肌组织总NO(NOx)含量测定:
采用比色法测定,具体操作按照试剂盒说明书进行。
结果
1.β_3AR细胞外第二环肽段主动免疫模型的建立:
首次免疫后2周,β_3AR免疫组大鼠血清中β_3AR自身抗体水平(2.08±0.18)已明显升高,与免疫前(0.28±0.03)及同期对照组(0.33±0.08)相比差异均具有统计学意义(P＜0.01),随后抗体水平缓慢升高并维持在一个高水平。对照组大鼠血清中β_3AR自身抗体水平始终无明显改变(P＞0.05)。(见图11)提示主动免疫模型制备成功。
2.主动免疫过程中大鼠心功能的变化:
初期免疫后3月及6月,β_3AR免疫组大鼠在体左心室功能参数LVSP和±dp/dt_(max)均高于对照组,而LVEDP低于对照组,但这些差异均无显著性(见表6)。提示β_3AR自身抗体的持续作用并未造成实验大鼠心功能的明显改变,但有改善心功能的倾向。
3.主动免疫过程中大鼠心肌形态学改变特点:
β_3AR免疫组大鼠在初期免疫后3月及6月,通过染色技术结合光学显微镜技术证实,心肌组织形态基本正常,未出现心肌细胞肥大、变性、坏死、大量炎性细胞浸润、胶原纤维化等病理改变;通过电子显微镜技术观察到β_3AR免疫组大鼠心肌细胞肌原纤维排列整齐,线粒体结构清晰完整(见图12)。提示在主动免疫6个月的情况下,β_3AR自身抗体的持续作用并未造成实验大鼠心肌结构发生明显改变。这提示需要进一步延长免疫时间或建立被动免疫心衰模型大鼠,以便最终确定β_3AR自身抗体在心衰发生发展过程中的作用机制。
4.主动免疫过程中大鼠心肌组织β_3AR的含量:
在免疫3月及6月,β_3AR免疫组大鼠心肌细胞膜β_3AR的含量均稍有增加,但与同期免疫对照组大鼠相比并无明显差异(P＞0.05)(见图13)。提示β_3AR自身抗体持续作用6个月对大鼠心脏β_3AR的含量未产生明显影响。
5.主动免疫过程中大鼠心肌组织eNOS的含量:
与免疫对照组相比,β_3AR免疫组大鼠心肌组织eNOS的含量在免疫3月及免疫6月时均有显著增加(P＜0.05)(见图14)。提示eNOS可能是介导β_3AR自身抗体作用的一个信号通路分子。
6.主动免疫过程中大鼠心肌组织cAMP和cGMP的含量:
与免疫对照组组相比,免疫3月时β_3AR免疫组大鼠心肌组织cGMP的含量有明显升高(25.35±1.10 vs 21.10±1.33 pmol/g,P＜0.05);免疫6月时cGMP含量升高更为显著(26.97±1.05 vs 20.45±1.58 pmol/g,P＜0.01)(见图15)。同期β_3AR免疫大鼠心肌组织cAMP的含量与免疫对照组组相比也有升高的趋势,但无显著差异(P＞0.05)(见图16)。进一步比较不同免疫时期大鼠心肌组织cAMP/cGMP的比值,发现此值在β_3AR免疫组大鼠免疫6月时明显低于免疫3月(5.38±0.22 vs 6.59±0.16,P＜0.05)而相应免疫对照组却无明显改变(6.71±0.17 vs 6.80±0.37,P＞0.05)(见图17)。说明在长期β_3AR免疫过程中心肌组织cGMP较cAMP确实有显著升高。提示cGMP可能是介导β_3AR自身抗体作用的一个重要信号通路分子。
7.主动免疫过程中大鼠心肌组织总NO(NOx)含量:
与免疫对照组组相比,免疫3月及6月时β_3AR免疫组大鼠心肌组织NOx的含量均有所升高,但未具有显著性差异(P＞0.05),(图18)提示β_3AR自身抗体可能刺激了NO产生。未出现显著意义的原因也许是由于本次实验为初探性研究,实验中所用动物数量较少造成的。
小结
1.在用β_3AR细胞外第二环肽段主动免疫6个月的条件下,高水平β_3AR抗体较长时间的存在,对大鼠心脏功能及结构均未产生有统计学显著意义的影响,但有改善心功能的倾向;
2.在主动免疫过程中,大鼠心肌组织β_3AR的含量均有增加的倾向,但未达到有统计学显著意义的水平:
3.β_3AR抗体存在时,大鼠心肌细胞中eNOS-NO-cGMP信号转导途径中的主要信号分子均有明显增加。提示β_3AR自身抗体可能通过eNOS-NO-cGMP信号转导途径发挥其病理生理作用。
结论
1.在不同原发疾病所致心衰患者及正常人血清中均存在有分别针对β_3AR细胞外第一环、第二环和第三环的自身抗体,但其中以针对β_3AR细胞外第二环的自身抗体(anti-β_3-EC_Ⅱ)出现的阳性率和抗体水平为最高(显著高于一、三环的自身抗体),且在心衰病人显著高于正常人,提示anti-β_3-EC_Ⅱ是重要的功能性抗体,参与了心衰(主要是扩心病和冠心病所致心衰)的病理生理过程,可能对由β_1AR自身抗体诱发的心脏功能障碍和心肌损伤起着心肌保护的作用。
2.心衰大鼠模型在心衰发生发展过程中产生了β_3AR自身抗体,且该抗体具有一定的产生、维持和减退的自然消长过程;β_3AR自身抗体的产生过程与脾淋巴细胞抗体生成能力增高的趋势一致,而与B淋巴细胞数量无关;伴随β_3AR自身抗体的增高,心肌组织β_3AR的数目也有上调,说明二者在功能上的一致性,高度提示其在心衰中具有一定的作用;
3.在主动免疫6个月条件下,β_3AR自身抗体对在体大鼠的持续作用未能引发心脏结构和功能的明显改变,但有改善心功能的倾向:此外,β_3AR抗体长期存在,可以使大鼠心肌组织中eNOS-NO-cGMP信号转导途径活跃,提示β_3AR自身抗体可能通过该途径发挥其病理生理作用。
Autoimmune mechanisms have been proposed to participate in the pathogenesis of chronic heart failure(CHF).In this study,we examined the immunoglobulin fractions of sera from healthy subjects and patients with chronic heart failure suffering from dilated cardiomyopathy (DCM),coronary artery disease(CAD),rheumatic heart disease(RHD) and hypertensive heart disease(HHD) for the presence of autoantibodies against the three extracellular loops ofβ_3-adrenoceptor.Autoantibodies against the three extracellular loops ofβ_3-adrenoceptor(anti-β_3-EC_Ⅰ,anti-β_3-EC_Ⅱand anti-β_3-EC_Ⅲ) were all detected in the sera of patients with heart failure and healthy subjects.However,among them the occurrence frequency and level of anti-β_3-EC_Ⅱwere the highest and by far higher than those of anti-β_3-EC_Ⅰand anti-β_3-EC_Ⅲ.In addition,the frequency and level of anti-β_3-EC_Ⅱin patients with heart failure(resulted mainly from DCM and CAD) were significantly higher than those in healthy subjects.Consequently,it is rational to assume that anti-β_3-EC_Ⅱis an important functional antibody involved in the pathogenesis of heart failure.The frequency of anti-β_3-EC_Ⅰin patients with DCM was also higher than that in healthy subjects but with fairly low titre level,while no statistical differences in both frequency and level of anti-β_3-EC_Ⅲwere seen between patients with CHF and healthy subjects.The effects of anti-β_3-EC_Ⅱon the cardiac function level of the failing heart were also analyzed.It was suggested that anti-β_3-EC_Ⅱmight play a functional protective role in the development of heart failure.
Experimental rat models with aortic banding were used to study the relations of development of heart failure to the genesis of serum autoantibodies to cardiacβ_3-adrenoceptor. 120 Wistar rats were divided into 2 groups:aortic banding group(n=80) and sham operated control group(n=40).On the basis of cardiac anatomic measurements,light microscopic examination,as well as myocardial collagen staining,it was shown that the experimental models underwent remarkable myocardial remodeling.During this period,the level of the autoantibodies againstβ_3-adrenoceptor in experimental group was exceedingly increased as compared with pre-treatment level accompanied by increased expression of cardiacβ_3-adrenoceptor protein, while in control group no significant changes were observed.These results suggest that the up-regulation of myocardialβ_3AR might be a crucial factor involving in the stimulation of genesis ofβ_3AR autoantibody during the course of heart failure.Moreover,the antoantibody againstβ_3AR exhibited the time course of occurrence,maintenance and disappearance,which is consistent with the trends of antibody producing ability alternation,confirming that the induction ofβ_3AR autoantoantibody is independent on the quantity of B lymphocytes,but is mainly regulated by functional statues of immune system.This might be an important form that immunological mechanism involved in the pathophysiological mechanisms of some caridac diseases.Furthermore,the mechanisms ofβ_3AR autoantoantibody need to be explored in the future.
An experiment model of cardiomyopathy was created by immunizing rats for 6 months with synthetic peptides corresponding to the sequence of the second extracellular loop of bothβ_3-adrenoceptor.Twenty rats were used and divided into two groups:a control group(n=8) and a group immunized with the peptide corresponding to theβ_3-adrenoceptor(β_3 immunity group, n=12).In the sera fromβ_3 immunized rats,high-level of anti-β_3-adrenoceptor antibodies were found throughout the study period,but noly a low level of antibodies were found in the sera from some control rats or in the preimmune sera of some immunized rats.At 3 and 6 months after immunization,the left ventricular systolic and diastolic function in immunized group were slightly but not significantly enhanced than that of in the control group,and morphological examination of the hearts ofβ_3 immunized rats demonstrated no obvious alterations.Moreover, compared with the corresponding controls,the myocardialβ_3-adrenoceptor density and the concentration of myocardial total NO of immunized rats displayed a slightly up-regulation and the myocardial eNOS and cGMP concentrations of immunized rats displayed significantly increased after 3 and 6 months of immunization.These results suggested that tha autoantibodies againstβ_3-adrenoceptor maybe transducte signal through eNOS-NO-cGMP pathway,however, immunization by peptide corresponding to the target sequences for anti-β_3-adrenoceptor antibodies for 6 months did not induces obviously morphological and functional changed in the heart.
引文
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