异种及同种异体间充质干细胞免疫排斥及细胞免疫调节作用研究

Immune rejection and cellular immune regulatory effects on the heterogeneous and allogeneic mesenchymal stem cells

目的观察异种及同种异体间充质干细胞(MSC)移植所致的免疫排斥反应,及其对异基因T细胞活化增殖的细胞免疫调节作用。方法分离、纯化人脐带间充质干细胞(h UC-MSC)及小鼠骨髓间充质干细胞(m BM-MSC),经流式细胞仪鉴定其表面标志物。在体实验:取20只无特定病原体级健康雌性BALB/C小鼠,随机分成h UC-MSC输注组和m BM-MSC输注组(分别于第1、4和15天经尾静脉注射2×106个相应的MSC),溶剂对照组(于相同时间点经尾静脉注射等体积质量分数为0.90%的氯化钠溶液)和空白对照组(不作任何处理),每组5只;上述处理结束后,继续饲养15 d,观察小鼠急性免疫排斥反应情况。离体实验:取初始T细胞或活化T细胞作为实验细胞,分为对照组、h UC-MSC组和m BM-MSC组,后2组以相应的MSC和T细胞共培养,对照组不加MSC培养;观察MSC对T细胞的细胞因子分泌、表面活化标志及增殖的影响。结果小鼠经多次尾静脉移植h UC-MSC及m BM-MSC后均未出现明显急性免疫排斥反应。初始T细胞离体实验中,h UC-MSC组、m BM-MSC组分别与对照组比较,T细胞培养上清中白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)水平,T细胞的早、中和晚期活化标志物分化抗原(CD)69、CD25和CD71的表达水平以及T细胞增殖率差异均无统计学意义(P>0.05)。活化T细胞离体实验中,h UC-MSC组、m BM-MSC组分别与对照组比较,T细胞培养上清中IL-2和IFN-γ水平均下降(P<0.01),T细胞的CD69、CD25和CD71的表达水平均下调(P<0.05),T细胞增殖率均下降(P<0.05)。结论 MSC不引起受体动物急性免疫排斥反应,不激活异基因T细胞,能抑制异基因T细胞活化与增殖,提示异种及同种异体MSC均无免疫原性,且能发挥细胞免疫调节作用。

Objective To investigate the immune rejection of heterogeneous and allogeneic mesenchymal stem cell（ MSC）transplantation in vivo and cellular immunoregulation effects of MSC on T lymphocyte in vitro. Methods Human umbilical cord mesenchymal stem cell（ h UC-MSC） and mouse bone marrow mesenchymal stem cell（ m BM-MSC） were isolated and purified,and their surface markers were identified by flow cytometry. Experiment in vivo： 20 specific pathogen free healthy female BALB / C mice were randomly divided into 4 groups： h UC-MSC grafted group,m BM-MSC grafted group,solvent control group and blank control group,with 5 mice in each group. About 2 × 106 MSC were intravenously injected through caudal vein into the two grafted groups on day 1,4 and 15. The solvent control group was injected with the same volume of0. 90%（ mass fraction） sodium chloride solution at the same time points. The blank control group was not treated. The acute immune rejection reactions were observed after 15 days of treatment. Experiment in vitro： mouse nave T cells and activated T cells were isolated. Nave T cells or activated T cells were divided into control group,h UC-MSC group and m BM-MSC group. MSC were co-cultured with T cells in h UC-MSC group and m BM-MSC group,while the control group was cultured without MSC. Effects of MSC on cytokines secretion,activation markers and proliferations of T cells were measured. Results No significant acute immune rejection was observed after h UC-MSC and m BM-MSC grafted repeatedly in mice. For nave T cells in vitro experiment,the interleukin-2（ IL-2） and interferon-γ（ IFN-γ） levels in T cells culture supernate,the expression levels of differentiation antigen CD69,CD25 and CD71 of T cells activation markers at early,medium and late stage,as well as the proliferation rate all showed no significant difference（ P〉0. 05） in the h UC-MSC and m BM-MSC groups,comparing to the control group. In the in vitro experiment for activated T cells,the IL-2 and IFN-γ levels in T cells culture supernate decreased（ P〈0. 01）,the expression levels of CD69,CD25 and CD71 all decreased（ P〈0. 05）,the proliferation rate decreased（ P〈0. 05） in the h UC-MSC and m BM-MSC groups,compared to the control group.Conclusion MSC could not cause acute immune rejection and activate the heterogenous T cells of transplant recipient animals. However,MSC could inhibit allogeneic T cells activation and proliferation. In conclusion,both heterogeneous and allogeneic MSC have no immunogenicity,and could regulate cellular immune reaction.