摘要： 目的 探讨骨髓间充质干细胞对实验性自身免疫性重症肌无力大鼠模型的治疗作用及其可能机制。方法 全骨髓贴壁筛选法分离培养Lewis大鼠骨髓间充质干细胞，流式细胞术检测其表面分子CD34、CD44 的表达。经Lewis 大鼠尾静脉注射经体外培养至第3 代的骨髓间充质干细胞，7 d 后腹腔注射抗乙酰胆碱受体单克隆抗体（mAb35）建立实验性自身免疫性重症肌无力动物模型，按照Lennon 评分标准进行临床症状评分、Western blotting 法检测肌肉组织中乙酰胆碱受体蛋白表达、流式细胞术检测外周血CD4+CD25+ T 细胞比率、酶联免疫吸附试验检测血清转化生长因子-β、干扰素-γ和IL-4 表达变化。结果 与生理盐水对照组相比，注射骨髓间充质干细胞的实验性自身免疫性重症肌无力大鼠模型临床评分降低（t = 8.062，P = 0.000），乙酰胆碱受体α蛋白表达水平升高（t = 8.092，P = 0.000）；同时血清转化生长因子-β表达水平升高（t = 9.859，P = 0.000），干扰素-γ（t = 9.040，P = 0.000）和IL-4（t = 3.320，P =0.004）表达水平降低，两组之间差异均有统学意义（P < 0.05）。结论 骨髓间充质干细胞能够显著减轻实验性自身免疫性重症肌无力大鼠模型临床症状，减少乙酰胆碱受体缺失。其作用机制可能与分泌免疫抑制性细胞因子转化生长因子-β上调CD4+CD25+调节性T 细胞表达水平，以及抑制炎性促进因子干扰素-γ和IL-4的分泌有关。

关键词: 重症肌无力, 自身免疫性, 实验, 间质干细胞移植, 印迹法, 蛋白质, 骨髓移植, 流式细胞术

Abstract: Objective To investigate the effect of bone marrow stem cells (BMSCs) on the treatment of experimental autoimmune myasthenia gravis (EAMG) and the possible pathogenesis. Methods BMSCs were obtained from Lewis rat bone marrow selected by cell attachment, cultured and proliferated in vitro. The expression of surface molecules of CD34 and CD44 was analyzed by flow cytometry. The Lewis rats were divided randomly into group A and group B. The rats in group A were transplanted with the third passage BMSCs, and the rats in group B were injected with physiological saline. Seven days after injection, the rat model of EAMG was established by injecting mAb35 to rats in both groups. Clinical signs and symptoms were assessed with Lennon score. Western blotting method was used to determine the expression of acetylcholine receptor (AChR) protein. The CD4+CD25+ Treg cell ratio was analyzed by flow cytometry. Plasma TGF-β, IFN-γ and IL-4 expressions were detected by ELISA. Results The clinical scores of rats treated with BMSCs were significantly lower than those untreated (t = 8.062, P = 0.000). In comparison with group B, the expression levels of AChR protein (t = 8.092, P = 0.000) and TGF-β (t = 9.859, P = 0.000) in group A were all higher, while IFN-γ (t = 9.040, P = 0.000) and IL-4 (t = 3.320, P = 0.004) were all lower in group A. Conclusion Transplantation with BMSCs can relieve the clinical syndrome of EAMG and reduce the lost of AChR by promoting the level of TGF-β, up regulating the expression of CD4+CD25+ Treg cell ratio, and suppressing IFN-γ and IL-4.

Key words: Myasthenia gravis, autoimmune, experimental, Mesenchymal stem cell transplantation, Blotting, western, Bone marrow transplantation, Flow cytometry