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目的了解辅助性T细胞Th1、Th2和Th17亚群在自身免疫性心肌炎的反应状态,从而探讨该病的免疫学发病机制。方法心肌C蛋白和完全弗氏佐剂足底注射、百日咳毒素腹腔内注射制作实验性自身免疫性心肌炎(EAM)大鼠模型。未给予免疫注射正常大鼠作为对照。在免疫注射后1周、2周、4周、6周和8周,经胸行心脏超声心动图检测。取心脏,行苏木素-伊红染色确定心肌炎症积分,天狼星红染色确定心肌纤维化。采用细胞内细胞因子染色和四色荧光抗体流式细胞术检测各组大鼠脾细胞中Th1、Th2和Th17比例,酶联免疫吸附试验检测外周血Th1相关细胞因子干扰素(IFN-)γ、Th2相关细胞因子白细胞介素(IL-)4和Th17相关细胞因子IL-17水平。结果经胸超声心动图显示EAM大鼠至免疫注射后第4周心脏射血分数维持正常值,左室收缩末期直径和左室舒张末期直径缩小;至第8周心脏射血分数明显降低,左室收缩末期直径和左室舒张末期直径增加。炎性积分在免疫注射后2周上调,4周达高峰,6周时逐渐降低;心肌纤维含量在第1周和第2周无明显改变,至第4周明显增强并达高峰,持续至第8周。脾细胞Th1及其相关细胞因子IFN-γ第1周反应血清水平升高,第4周达高峰,第6周开始下降。Th17及IL-17在第2～4周升高,水平维持至第6～8周。Th2在前4周无明显变化,IL-4在第4周开始升高,两者均在第6周迅速升高,并持续至第8周。结论在EAM大鼠模型中,2～4周是心肌炎症阶段,4～8周是纤维化阶段。在此过程中出现Th1/Th2失衡,其中炎症性心肌炎阶段以Th1、Th17亚群介导的细胞免疫为优势,纤维化心肌炎阶段以Th2和Th17亚群介导的体液免疫为优势,Th17衔接机体细胞免疫和体液免疫。
Objective To investigate the response of Th1, Th2 and Th17 helper T cells in autoimmune myocarditis and to explore the immunological pathogenesis of this disease. Methods Experimental myocardial autoimmune myocarditis (EAM) rat model was made by intraperitoneal injection of cardiac C protein and complete Freund’s adjuvant and intraperitoneal injection of pertussis toxin. No immunized normal rats were given as control. One week, two weeks, four weeks, six weeks and eight weeks after immunization were performed by transthoracic echocardiography. The heart was harvested and the myocardial inflammation scores were determined by hematoxylin-eosin staining and myocardial fibrosis was determined by Sirius red staining. The ratio of Th1, Th2 and Th17 in splenocytes of each group was detected by intracellular cytokine staining and four-color fluorescent antibody flow cytometry. Th1-related cytokine interferon (IFN-) γ in peripheral blood was detected by enzyme linked immunosorbent assay (ELISA) Th2-related cytokines interleukin (IL-) 4 and Th17-associated cytokine IL-17 levels. The results of transthoracic echocardiography showed that EAM rats maintained normal values of ejection fraction, diameter of left ventricular end-systole and diameter of left ventricular end-diastole in 4 weeks after immunization. By the 8th week, the ejection fraction of left End-systolic diameter and left ventricular end diastolic diameter increased. Inflammatory scores increased at 2 weeks after immunization, peaked at 4 weeks, and gradually decreased at 6 weeks. The content of myocardial fiber did not change significantly at 1 week and 2 weeks, and significantly increased and reached the peak at 4 weeks, 8 weeks. Serum levels of Th1 and its related cytokine IFN-γ in the first week of response increased, peaked at the fourth week, and began to decline at the sixth week. Th17 and IL-17 in the second to 4 weeks increased, the level is maintained until the 6th to 8 weeks. Th2 had no significant change in the first 4 weeks, IL-4 began to rise in the 4th week, and both of them rose rapidly in the 6th week and continued to the 8th week. Conclusions In the EAM rat model, 2 to 4 weeks are stages of myocardial inflammation and 4 to 8 weeks are stages of fibrosis. In this process, Th1 / Th2 imbalance occurs. Inflammatory myocarditis is dominated by cellular immunity mediated by Th1 and Th17 subtypes, while humoral immunity mediated by Th2 and Th17 subtypes is predominant in fibrotic myocarditis. Th17 adapter Cellular immunity and humoral immunity.