Th1/Th9/Th17细胞失衡对实验性自身免疫性重症肌无力幼鼠的影响及机制初探

Effects of Th1/Th9/Th17 cells imbalance on young rats with experimental autoimmune myasthenia gravis and its mechanism

目的:对实验性自身免疫性重症肌无力(myasthenia gravis,MG)幼鼠采用双类似物(Lys262-Ala207)经鼻黏膜诱导免疫耐受后,了解实验幼鼠Th1/Th17/Th9细胞及产生相关细胞因子的细胞变化情况及其与EAMG幼鼠发病的相关性,探讨其调控作用机制。方法:将30只幼年雌性C57BL/6小鼠采用完全随机分组方法分为3组:模型组、耐受组和对照组。模型组经腹腔注射含1.0 mg/kg mAb35的Ringer’s液0.2 ml,建立EAMG模型;耐受组在致敏前10 d先经鼻黏膜滴入Lys262-Ala207诱导黏膜耐受,再按照模型组方式进行处理;对照组注射不含mAb35的Ringer’s缓冲液0.2 ml。采用流式细胞仪检测小鼠脾脏细胞中分别代表Th1/Th17/Th9细胞功能的CD4+干扰素(interferon,IFN)-γ+细胞、CD4+白介素(interleukin,IL)-17+细胞和CD4+IL-9+细胞的含量。采用双抗体夹心ELISA法检测脾细胞培养上清中IFN-γ、IL-17、IL-9的水平。结果:(1)MG的临床表现对照组明显轻于模型组和耐受组,而耐受组经双类似物免疫耐受后,其临床症状也较模型组明显减轻。(2)模型组、耐受组和对照组的CD4+IFN-γ+细胞含量3组间比较差异有统计学意义(P=0.00),即:模型组分别比耐受组和对照组高(P=0.00),而耐受组也比对照组高(P=0.00);(3)模型组、耐受组和对照组的CD4+IL-17+细胞含量3组间比较差异有统计学意义(P=0.00),即:模型组分别比耐受组和对照组高(P=0.00),而耐受组也比对照组高(P=0.00);(4)模型组、耐受组和对照组的CD4+IL-9+细胞含量3组间比较差异有统计学意义(P=0.00),即:模型组分别比耐受组和对照组高(P=0.00),而耐受组也比对照组高(P=0.00)。(5)模型组、耐受组和对照组的脾细胞上清中细胞因子IFN-γ、IL-17、IL-9的水平3组间比较差异有统计学意义(P=0.00),即:模型组分别比耐受组和对照组高(P=0.00),而耐受组也比对照组高(P=0.00)。结论:采用Lys262-Ala207对实验幼鼠进行鼻黏膜耐受后不仅能有效的缓解其MG的临床症状,同时还能下调导致实验幼鼠发病的CD4+IFN-γ+细胞、CD4+IL-17+细胞和CD4+IL-9+细胞含量水平以及细胞因子IFN-γ、IL-17、IL-9的水平,缓解Th1/Th17/Th9细胞功能失衡现象,从而达到预防和减轻实验幼鼠发病的作用。

Objective ：To study the variation of Thl/Thl7/Th9 ceils which produce cytokines and to explore the correlation between these cells and pathogenesis of experimental autoimmune myasthenia gravis （EAMG） young mice after being treated with dual analogue（Lys262-Ala207） to create immune tolerance effect through nasal mucosa. Methods：Thirty young C57BL/6 female rats were randomized into three groups：model group,tolerance group and control group. Rats in model group were injected with 0.2 ml Ringer＇s containing mAb35（ 1.0 mg/kg） to establish EAMG model;rats in tolerance group were given Lys262-Ala207 intranasally for ten successive days before immunization then were injected 0.2 ml Ringer＇s containing mAb35（1.0 mg/kg）;rats in control group were intraperi-toneally injected 0.2 ml Ringer＇s without containing mAb35 （1.0 mg/kg）. Cells of CD4＋ IFN-γ＋, CD4＋ IL-17＋ and CD4＋ IL-9＋, which respectively represent the cells of Thl/Th17/Th9 in spleen cell of young rats were detected by flow cytometry. IFN- γ,IL-17 and IL-9 in supernatant of spleen cell were detected by Antibody-sandwich ELISA. Results：（1）Clinical symptom of myas- thenia was less obvious in control group than in model group and tolerance group and clinical symptom of myasthenia was alleviated in tolerance group than in model group after being given dual analogue. （2）There were significant differences in the content of CD4＋ IFN-γ＋ among three groups （model group, tolerance group and control group）（P=0.00） ;content of CD4＋ IFN-γ＋ was higher in model group than in the other groups （P=0.00） and content of CD4~ IFN-γ＋ was higher in tolerance group than in control group （P=0.00）. （3） There were significant differences in the content of CD4＋ IL-17＋ among three groups （model group, tolerance group and control group） （P=-0.00） ;content of C D4＋ IL-17＋ was higher in model group than in the other groups （P=0.00） and content of C D4＋ IL-17＋ was higher in tolerance group than in control group（P=0.00）. （4）There were significant differences in the content of CD4＋ IL-9＋ among three groups（model group, tolerance group and control group）（P=0.00） ;content of CD4＋ IL-9＋ was higher in model group than in the other groups（P=0.00） and content of CD4＋ IL-9＋ was higher in tolerance group than in control group（P=-0.00）. （5）There were signifi- cant differences in the content of IFN-γ, IL-17 and IL-9 in supernatant of spleen cell among three groups（model group,toler- ance group and control group）（P=0.00） indicating that the content was higher in model group than in the other groups（P=0.00） and the content was higher in tolerance group than in control group（P=-0.00）. Conclusions：Clinical symptom of myasthenia is relieved ef- fectively, content of CD4＋ IFN-γ＋, CD4＋ IL-17＋ and CD4＋ IL-9＋ as well as the content of IFN-γ,IL-17 and IL-9 which lead to sickness of rats is reduced and unbalance of cell of Thl/Yhl7/Th9 is relieved after young rats being treated with dual analogue（Lys262-Ala207） to create immune tolerance through nasal mucosa. This clarifies the role of dual analogue in the mechanism of immune tolerance of young rats.