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目的探讨大鼠骨髓间充质干细胞(BMSCs)异体移植对小鼠实验性自身免疫性脑脊髓炎(EAE)的治疗作用。方法全骨髓贴壁培养法获得大鼠BMSCs,流式细胞术检测细胞免疫表型,并诱导成骨方向分化。取雌性C57BL/6小鼠,随机分为3组:正常对照组、PBS组和大鼠BMSCs组,用髓鞘少突胶质细胞糖蛋白(MOG)35~55联合完全弗氏佐剂诱导建立EAE模型。小鼠免疫后38d和48d腹腔注射PBS或大鼠BMSCs进行治疗,神经功能评分观察各组小鼠神经功能变化。二次治疗12d后取各组小鼠脊髓、脾脏和外周血。HE染色及Luxol fast blue染色观察脊髓炎性细胞浸润及髓鞘脱失情况;脾脏制成单细胞悬液,细胞CFSE标记后10mg/L刀豆球蛋白(Con A)和MOG_(35~55)刺激培养3d,观察脾细胞增殖情况。ELISA检测大鼠BMSCs移植后外周血细胞因子干扰素γ(IFN-γ)、白细胞介素17(IL-17)含量。结果流式细胞术显示,大鼠BMSCs第3代(P3)细胞表达抗原CD29、CD90、CD106,不表达CD45。体外诱导其能向成骨分化。小鼠发病后,大鼠BMSCs组小鼠神经功能缺损症状较PBS组减轻,评分降低。HE染色和Luxol fast blue染色结果显示,大鼠BMSCs组脊髓炎细胞浸润和脱髓鞘比同时间点PBS组减轻(P<0.05)。Con A和MOG_(35~55)刺激培养后,PBS组和大鼠BMSCs组脾细胞增殖增加,而大鼠BMSCs组又较PBS组降低。与PBS组相比,大鼠BMSCs组血浆细胞因子IFN-γ、IL-17含量降低(P<0.05)。结论全骨髓贴壁培养法能有效分离纯化BMSCs,大鼠BMSCs异体移植对小鼠EAE模型有治疗作用。
Objective To investigate the therapeutic effect of allogeneic transplantation of rat bone marrow mesenchymal stem cells (BMSCs) on experimental autoimmune encephalomyelitis (EAE) in mice. Methods BMSCs were obtained from whole bone marrow adherent culture. Cell immunophenotype was detected by flow cytometry and osteogenic differentiation was induced. Female C57BL / 6 mice were randomly divided into 3 groups: normal control group, PBS group and BMSCs group, induced by myelin oligodendrocyte glycoprotein (MOG) 35-55 combined with complete Freund’s adjuvant EAE model. The mice were injected intraperitoneally with PBS or BMSCs at 38 and 48 days after immunization. The neurological function scores of the mice in each group were observed. After 12 days of secondary treatment, the spinal cord, spleen and peripheral blood of each group were taken. HE staining and Luxol fast blue staining were used to observe inflammatory cell infiltration and demyelination of spinal cord. The spleen was made into a single cell suspension. After the cells were labeled with CFSE, 10 mg / L concanavalin A (Con A) and MOG 35-55 Stimulate the culture 3d, observe the proliferation of spleen cells. The levels of IFN-γ and IL-17 in peripheral blood were measured by ELISA. Results Flow cytometry showed that the 3rd generation (P3) cells of BMSCs of rats expressed antigens CD29, CD90, CD106 and did not express CD45. In vitro induction of osteogenic differentiation. After the onset of the mice, the symptoms of neurological deficits in the BMSCs of rats were relieved and the scores were decreased. The results of HE staining and Luxol fast blue staining showed that the infiltration and demyelination of myelitis cells in the BMSCs group were relieved at the same time point (P <0.05). Con A and MOG_ (35-55) stimulated culture, splenocytes proliferation increased in PBS group and BMSCs group, but decreased in BMSCs group and PBS group. Compared with PBS group, the levels of plasma cytokines IFN-γ and IL-17 in BMSCs of rats decreased (P <0.05). Conclusion All bone marrow adherent culture can effectively separate and purify BMSCs. Allogeneic transplantation of rat BMSCs has a therapeutic effect on EAE model in mice.