全反式维甲酸对乙酰胆碱受体特异性淋巴细胞的免疫调控作用

The Immunomodulatory Effects of All-trans Retinoic Acid on AChR-specific Lymphocytes

目的:观察全反式维甲酸(ATRA)对乙酰胆碱受体(AChR)特异性淋巴细胞的体外调控作用,探讨其治疗重症肌无力(MG)的可能机制。方法:建立完全弗氏佐剂(CFA)对照组及实验性自身免疫性重症肌无力(EAMG)组大鼠,并获取淋巴结单个细胞悬液,以ACh R97-116多肽片段以及不同浓度的ATRA体外培养72 h,采用流式细胞仪法、CCK-8法、ELISA法分别检测活细胞比例、细胞凋亡和周期的改变以及Th亚群的格局和B细胞抗体分泌能力的变化。结果:ATRA显著降低活细胞比例(P<0.001);不同浓度的ATRA均促进了特异性细胞群的凋亡(P<0.001),且呈剂量依赖性,而ATRA未改变AChR特异性淋巴细胞的生长周期;ATRA处理后,CFA和EAMG组的淋巴细胞增殖均受到明显抑制,且ATRA对ACh R特异性的淋巴细胞的抑制明显(EAMG组,P<0.01)于CFA组(P<0.05);ATRA干预后,ACh R特异性CD4+T淋巴细胞的比例下降(P<0.01),且ATRA促进了Th2、Treg细胞亚群百分比(P_(IL-4)<0.001,P_(Foxp3)<0.001),而抑制了促炎性的Th17、Th1细胞亚群百分比(P_(IL-17)<0.05,P_(IFN-γ)<0.001);ATRA能够降低ACh R特异性B细胞的抗体分泌能力(P<0.01)。结论:ATRA不仅能抑制ACh R特异性T细胞功能,同时也能抑制ACh R特异性B细胞功能,其在MG的临床治疗中可能起治疗作用。

Objective： To observe the effects ofAll-trans retinoic acid （ATRA） on the immune functions of AChR-specific lymphcytes via in vitro assays, and investigate the possibility of ATRA in the clinical treatment of myasthenia gravis （MG）. Methods： CFA control group and EAMG experimental rats were established to obtain single lymphocytes suspension and cells were followed by AChR97-116 peptide with or without ATRA stimulation for 72 h, and then viable cell population, cell apoptosis, cell cycle and the distri- bution of Th cells were determined by flow cytometry. CCK-8 assay was selected to evaluate the effects of ATRA on proliferatory ability of lymphocytes. ELISA was used to detect the antibody secretion orB cells affected by ATRA. Results： Compared with CFA group, lymphocytes obtained from EAMG rats had higher ratios of living cells, and this ratio was obviously decreased after ATRA treatment, P〈0. 001. Different concentrations of ATRA promoted the apoptosis of AChR-specific cells （P〈0.001）, and the promoted effects were ATRA dose-dependent, however, cell cycles were not changed. ATRA markedly inhibited the proliferation of cells from both CFA and EAMG groups, moreover, AChR-specific cells were more sensitive to ATRA treatment （P〈0.01） than that of cells from CFA rats （P〈0.05）. Thc ratio of AChR-specific CD4＋T cells was reduced by ATRA （P〈0.01）, and ATRA incubation significantly promoted the percentages of Th2, （PCD4＋IL4＋〈0.001）, Treg （PCD4＋Foxp3＋〈0.001） cell types, but markedly inhibited the percentages of Th17 （PCD4＋IL17＋〈0.05）, Thl （PCD4＋IFEγ＋〈0.001） cells. ELISA data showed us that ATRA obviously down regulated the antibody secretion of AChR-specific B cells, P〈0.01. Conclusions： ATRA not only inhibited the functions of AChR-specific T cells, but also suppressed the roles of AChR-specific B cells, predicating a therapeutic effect of ATRA on myasthenia gravis therapy.