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目的探讨重症肌无力(MG)患者外周血中Th17细胞及相关细胞因子白细胞介素17(IL-17)在MG发病中的作用。方法收集40例MG患者和10名健康人(对照组)外周血标本,采用流式细胞术检测外周血单个核细胞(PBMCs)中Th17细胞比例,反转录酶-聚合酶链锁反应(RT-PCR)检测PBMCs中维甲酸受体相关孤儿受体γt(RORγt)mRNA水平,ELISA检测血清中IL-17水平,放射免疫沉淀法检测血清中抗乙酰胆碱受体抗体(AChR-Ab)滴度;分离PBMCs中CD4~+T细胞和CD19~+B细胞与金黄色葡萄球菌肠毒素B(SEB)进行共培养,培养系统中加入人IL-17和(或)IL-21中和抗体,放射免疫测定法检测培养液中AChR-Ab滴度。采用MG评分(quantitative MG scoring system,QMGs)对MG的严重程度进行评估,并对MG患者的Th17细胞比例、RORγt mRNA和IL-17水平与病情QMGs的相关性,以及MG患者抗AChR-Ab滴度与PBMCs中Th17细胞比例的相关性进行分析。结果 MG患者PBMCs中Th17细胞比例[1.11%(0.90%,1.34%)]高于健康对照组Th17细胞比例[0.26%(0.08%,0.36%)](z=5.494,P<0.001),且与疾病严重程度呈正相关(r=0.4394,P=0.0046);血清中IL-17水平和PBMCs中RORγt mRNA相对表达[分别71.46(53.91,104.76)pg/mL、2.63(1.94,3.12)]均较健康对照组[分别18.82(12.73,29.80)pg/mL、1.13(0.98,1.28)]显著增高(均P<0.001);MG患者血清中抗AChR-Ab滴度[2.34(1.19,3.60)nmol/L]较健康对照组[-0.08(-0.24,-0.03)nmol/L]显著增高(z=4.662,P<0.001),且与Th17细胞比例呈正相关(r=0.7066,P=0.0001)。MG患者外周血T、B细胞与SEB共培养后抗AChR-Ab水平高于未加入SEB时及健康对照(均P<0.01);加入抗人IL-21或IL-17中和抗体后,两者AChR-Ab滴度与未加入抗体时AChR-Ab滴度比较均降低(均P<0.05),且均仍高于MG患者未加入SEB时及健康对照(P<0.01);在培养上清中同时加入抗人IL-21和IL-17中和抗体时AChR-Ab滴度明显低于加入单种抗体时,而与未加入SEB时及健康对照差异无统计学意义(均P>0.05)。结论 MG患者外周血中Th17细胞可能通过IL-17促进AChR-Ab产生,参与疾病的病理过程。
Objective To investigate the role of Th17 cells and related cytokine interleukin-17 (IL-17) in the pathogenesis of MG in patients with myasthenia gravis (MG). Methods Peripheral blood samples of 40 patients with MG and 10 healthy people (control group) were collected. The proportion of Th17 cells in peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. The ratio of RT - PCR -PCR was used to detect the mRNA level of RORγt in orphan receptor-related orphan receptor of PBMCs. ELISA was used to detect the level of IL-17 in serum, and the serum anti-acetylcholine receptor antibody (AChR-Ab) titer was detected by radioimmunoprecipitation. CD4 + T cells and CD19 + B cells in PBMCs were isolated and co-cultured with Staphylococcus aureus enterotoxin B (SEB). Human IL-17 and / or IL-21 neutralizing antibody was added to the culture system and radioimmunoassay Assay to detect AChR-Ab titers in culture broth. The severity of MG was evaluated by quantitative MG scoring system (QMGs). The correlations between Th17 cell ratio, RORγt mRNA and IL-17 levels and QMGs in patients with MG and AChR-Ab Degrees and the proportion of Th17 cells in PBMCs were analyzed. Results The proportion of Th17 cells [1.11% (0.90%, 1.34%)] in PBMCs of patients with MG was higher than that of healthy controls (0.26% (0.08%, 0.36%)] (R = 0.4394, P = 0.0046). The relative expression of IL-17 in serum and RORγt mRNA in PBMCs were 71.46 (53.91,104.76) pg / mL and 2.63 (1.94, 3.12) The serum levels of AChR-Ab in MG patients [2.34 (1.19,3.60) nmol / L, P <0.001) were significantly higher in the control group (18.82 vs. 12.73, 29.80 pg / mL and 1.13 ] (R = 0.7066, P = 0.0001), which was significantly higher than that in healthy controls [-0.08 (-0.24, -0.03) nmol / L] The level of anti-AChR-Ab in co-cultured T and B cells with peripheral blood of MG patients was significantly higher than that without SEB and healthy controls (all P <0.01). After anti-IL-21 or IL- The AChR-Ab titer was lower than that without antibody (all P <0.05), and both of them were still higher than those of MG patients without SEB and healthy controls (P <0.01) AChR-Ab titers were significantly lower than those of the control group (P> 0.05), but no difference was found between the two groups (P> 0.05) . Conclusion Th17 cells in peripheral blood of patients with MG may promote the production of AChR-Ab through IL-17 and participate in the pathological process of the disease.