重症肌无力被动转移幼鼠的黏膜免疫耐受研究

Nasal immune tolerance to passively transferred myasthenia gravis in young mice

目的观察特异性耐受原-双类似物（Lys262-Ala207）对重症肌无力被动转移（PTMG）小鼠模型的黏膜免疫效果，探讨其作用机理及该方法在模型应用中的可行性。方法将C57BL／6幼年雌性小鼠60只分为3组：耐受组、模型组、对照组，各20只。耐受组和模型组建立PTMG模型。耐受组在致敏前10d，每天经鼻腔滴入含耐受肽-双类似物25μg的PBS50μl；模型组连续10d经鼻腔滴入不含耐受肽的PBS50μl；正常对照组不做特殊处理。观察实验各组小鼠的体重、临床评分、白细胞介素4（IL-4）、γ干扰素（IFN-γ）、TGF-β1及其他各项指标的改变情况。结果模型组小鼠表现较为典型的PTMG症状；耐受组小鼠经耐受治疗有效，其临床症状较轻。血清乙酰胆碱受体抗体（AChRAb）含量耐受组为（16．01±1．09）mg／L，模型组为（28．12±1．28）mg／L，均高于对照组[（1．60±0．28）mg／L]（t=44．37，70．27，P〈0．01）。耐受组外周血中TGF-β1[（437．19±1．93）ng／L]高于模型组[（175．63±3．12）ng／L]（t=36．07，P〈0．01），模型组IL-4、IFN-γ[（193．37±3．95）ng／L，（320．46±2．14）ng／L]，均高于耐受组[（141．02±3．11）ng／L，（187．99±4．67）ng／L]（t=37．20、51．69，P〈0．01）。各因子与对照组比较仍未达正常水平，差异有统计学意义（t=26．65、31．05、49．02，P〈0．01）。结论双类似物鼻黏膜耐受对缓解PTMG有效，表现为：血清AChRAb含量降低，外周血中TGF-β1分泌增高，IL-4、IFN-γ分泌降低和临床症状的缓解等。同时该方法还具有使用方便、安全等特点。

Objective Young C57BL/6 （B6） mice were treated with a specific toleragen-dual analogue （ Lys262-Ala207 ） intranasally to observe its effect on the invasion process of mice model and the clinical symptoms, to assess its clinical effects, and to explore the underlying mechanisms and feasibility of nasal mucosal tolerance explored. Methods Passively transferred myasthenia gravis （PTMG） was induced by mAb35 on B6 young female mice. Sixty mice were divided equally into three groups： tolerance group, model group and control group. Lys262-Ala207 was given intranasally （250 μg/mouse） to tolerance group with mAb35 for 10 successive days before immunization. Model group received PBS 50 μl only. The body weight and clinical scores were evaluated. The serum levels of AChRAb and the main cytokines （ IL-4, IFN- γ, TGF-β1 ） were detected with ELISA. Results The model group had typical myasthenia symptoms. B6 mice of tolerance group had less severe symptoms compared with control groups. The clinical symptoms of tolerance group were relieved. The level of AChRAb in tolerance group [ （16.01 ± 1.09） mg/L] was significantly lower than that of model group [ （ 28. 12 ± 1.28 ） mg/L ] （ t=44. 37, P 〈 0. 01 ）. IL-4 and IFN-γ, levels in tolerance group [ （ 141.02 ± 3.11 ） ng/L, （ 187. 99 ± 4. 67 ） ng/L] were significantly lower than those of model group [ （ 193.37 ± 3.95） ng/L, （320. 46 ± 2. 14） ng/L] （ t = 37. 20, 51.69, P 〈 0. 01 ）. The level of TGF-β1 in tolerance group [ （437. 19 ± 1.93 ） ng/L ] was higher than that of model group [ （ 175.63 ± 3.12） ng/L] （ t = 36. 07 ,P 〈 0. 01 ）. But there were still significant change as compared to those in control group （t =26.65, 31.05, 49.02,P〈0.01）. Conclusions Nasal administration of Lys262-Ala207 ameliorated muscular weakness in PTMG young mice. The therapeutic effect is possibly correlated with the function of immune system.