骨髓间充质干细胞异体移植对小鼠实验性自身免疫性脑脊髓炎的治疗作用

doi:10.16098/j.issn.0529-1356.2017.03.004

摘要/Abstract

摘要：

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（ConA）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)。 ConA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。

关键词: 骨髓间充质干细胞, 异体移植, 实验性自身免疫性脑脊髓炎, 脾细胞增殖, 细胞因子, 流式细胞术, Luxol fast blue染色, 小鼠

Abstract:

Objective To investigate the therapeutic effect of allogeneic transplantation of rat bone marrow mesenchymal stem cell (BMSCs) on experimental autoimmune encephalomylitis (EAE) in mice. Methods Rat BMSCs were obtained by a whole-bone marrow adherent culture method and identified with flow cytometry for cellular immune phenotypes. The obtained rat BMSCs were induced to differentiate into osteoblasts. The female C57BL/6 mice were randomly divided into three groups: control group, PBS group and rat BMSCs group. The EAE was induced by myelin oligodendrocyte glycoprotein( MOG) _35-55 with fully emulsified Freund’s adjuvant and immunization by subcutaneous multi-point injection. The mice were treated with rat BMSCs by intraperitoneal injection 38 and 48 days after immunization. Neurological function scores were performed to evaluate the neurological function. Twelve days after the second treatment the spinal cord，spleen and peripheral blood were taken. HE staining and Luxol fast blue staining were used to observe inflammatory cell infiltration and demyelination in the spinal cord. Single-cell suspension was prepared and stimulated with 10mg/L concanavalin A (ConA) and MOG _35-55for 3 days. The splenocytes proliferation was observed. The peripheral blood interferon-γ（IFN-γ）、interleukin-17（IL-17）cytokines were evaluated by ELISA. Results Flow cytometry results showed that P3 cells of rat BMSCs expressed CD29, CD90 and CD106, but not expressed CD45. The obtained cells were induced to osteogenic cells. The neural function was less seriously damaged in the rat BMSCs group than the PBS group. HE staining indicated that the infiltration of inflammatory cells in rat BMSCs group was less than that in PBS group (P<0.05). Luxol fast blue staining displayed that the demyelination of rat BMSCs group was less than the PBS group at the same time point (P<0.05). After stimulation with ConA and MOG _35-55, the splenocytes proliferation of PBS group and rat BMSCs group was increased, while that of rat BMSCs group was lower than PBS group. Compared with PBS group, rat BMSCs group had decreased peripheral blood IFN-γ、IL-17 levels (P<0.05). Conclusion Rat BMSCs can be effectively isolated and purified by the whole bone marrow adherent culture method, and rat bone marrow mesenchymal stem cells have therapeutic effects on mouse EAE.

Key words: Bone marrow mesenchymal stem cell, Allograft, Experimental autoimmune encephalomylitis, Splenocyte proliferation, Cytokine；Flow cytometry, Luxol fast blue staining, Mouse

廖文萍胡蓉黄悦李红闫丽丽苏敏. 骨髓间充质干细胞异体移植对小鼠实验性自身免疫性脑脊髓炎的治疗作用[J]. 解剖学报, 2017, 48(3): 266-272.

LIAO Wen-ping HU Rong HUANG Yue LI Hong YAN Li-li SU Min. Therapeutic effect on mice experimental autoimmune encephalomyelitis of allogeneic bone marrow mesenchymal stem cell transplantation[J]. Acta Anatomica Sinica, 2017, 48(3): 266-272.

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