辅助性CD4^+ T细胞亚群失衡在实验性自身免疫性重症肌无力大鼠发病机制中的作用

Role of Unbalance of Helper CD4^+ T Lymphocyte Subsets in Pathogenesis of Experimental Autoimmune Myasthenia Gravis in Rats

目的研究实验性自身免疫性重症肌无力(Experimental autoimmune myasthenia gravis,EAMG)大鼠发病过程中辅助性CD4+T细胞亚型的变化。方法将Lewis大鼠分为完全弗氏佐剂(CFA)早、晚期发病时对照相和EAMG早期(初次免疫后13d)、高峰期(初次免疫后50d)发病时相4组,各组分别给予相应的药物后,于发病早期和高峰期分离淋巴细胞,采用流式细胞术检测各组大鼠淋巴细胞中Th1、Th2、Th17和Treg细胞的比例,ELISA法检测淋巴细胞培养上清中各种细胞因子水平。结果在EAMG发病的早期时相,与CFA组比较,EAMG组Th1、Th2、Th17细胞的比例下降,而Treg细胞比例上升;淋巴细胞培养上清中IFNγ和IL-4水平显著下降(P<0.05),IL-6水平显著升高(P<0.05),IL-17和TGF-β水平略升高,但差异无统计学意义(P>0.05)。在EAMG发病的高峰期,与CFA组比较,EAMG组Th1和Th17细胞的比例显著上升(P<0.05),而Th2和Treg细胞的比例显著下降(P<0.05),淋巴细胞培养上清中IFNγ和IL-17水平显著上升(P<0.05),IL-4和TGF-β水平显著下降(P<0.01),IL-6水平略上升,但差异无统计学意义(P>0.05)。结论在EAMG发生过程中,4种辅助性CD4+T细胞亚型之间的平衡被打破,晚期时相中Th1和Th17细胞比例上升,而Th2和Treg细胞比例下降。

Objective To study the change of helper CD4＋T lymphocyte subsets during pathogenesis of experimental autoimmune myasthenia gravis（EAMG）in rats.Methods Lewis rats were randomly divided into complete Freund adjuvant（CFA）and EAMG groups,eight for each.Each rat in EAMG group was primarily immunized s.c.with 200 μl of PBS containing 50 μg of RAChRα97-116,1 mg of tuberculin and 100 μl of incomplete Freund adjuvan（tIFA）,and boosted with 200 μl of PBS containing 50 μg of R-AChRα97-116 and 100 μl of IFA on day 30 after the primary immunization.However,each rat in CFA group was immunized by the same method with the PBS containing the above-mentioned components except R-AChRα97-116.The lymphocytes of rats in each group were isolated 13（early phase）and 50 d（peak phase）after primary immunization,in which the percentages of Th1,Th2,Th17 and Treg cells were determined by flow cytometry.The cytokine levels in culture supernatant of the lymphocytes were determined by ELISA.Results At early phase of EAMG,as compared with those in CFA group,the percentages of Th1,Th2,and Th17 cells decreased,while that of Treg cells increased;the IFNγ and IL-4 levels in culture supernatant of lymphocytes decreased significantly（P 0.05）,while the IL-6 level increased significantly（P 0.05）;the IL-17 and TGF-β levels increased slightly but showed no significant difference（P 0.05）.However,at peak phase of EAMG,as compared with those in CFA group,the percentages of Th1 and Th17 cells increased significantly（P 0.05）,while those of Th2 and Treg cells decreased significantly（P 0.05）;the IFNγ and IL-17 levels in culture supernatant of lymphocytes increased significantly（P 0.05）,while the IL-4 and TGF-β levels decreased significantly（P 0.01）;the IL-6 level increased slightly but showed no significant difference（P 0.05）.Conclusion During pathogenesis of EAMG,the balance of four helper T lymphocyte subsets was broken up.At late phase of EAMG,the percentages of Th17 and Th1 cells increased while those of Th2 and Treg cells decreased.