小鼠骨髓间充质干细胞对脾细胞免疫反应的影响

Effect of murine mesenchymal stem cells （mMSC） on the allogeneic immuno-response of syngeneic and allogeneic spleen cells

目的探讨小鼠骨髓间充质干细胞（mMSC）对脾细胞免疫反应性增殖的作用及机制。方法分别采用有丝分裂原刀豆球蛋白A（ConA）（20μg／ml）和异基因抗原（异基因小鼠脾细胞）作为刺激因素，观察不同比例mMSC对同基因和异基因脾细胞增殖的影响。MTT法测定脾细胞免疫反应性增殖水平；流式细胞术检测CD4^＋／CD8^＋细胞比值、CD4^＋CD25^＋细胞百分比；ELISA法检测IFN-γ、IL-2、TGF—β1、IL-4水平。结果①ConA刺激时，mMSC对同基因和异基因（BALB／c）脾细胞的反应性增殖的抑制作用相似，呈细胞数量依赖性，当细胞比例为1：1时，抑制率为84．21％。在异基因抗原刺激下，mMSC对同基因和异基因脾细胞反应性增殖的抑制作用相似，细胞比例为1：10时，抑制率为88．07％，呈细胞数量依赖性。②在ConA刺激下，mMSC—S脾细胞比例为1：1组的CD4^＋／CD8^＋比值为2．61±0．24，CD4^＋CD25^＋细胞比例为（6．34±0．69）％，与对照组比较差异有统计学意义（P〈0．05）。在异基因抗原刺激下，1：10组CD4^＋／CD8^＋比值为2．49±0．14、CD4^＋CD25^＋细胞比例为（8．31±0．80）％，与对照组比较差异有统计学意义（P〈0．05）。③在共培养体系中mMSC可抑制IFN-γ、IL-2的分泌，促进TGF—β1、IL-4的分泌，其水平与mMSC的数量呈正相关。结论在体外培养体系中，mMSC呈数量依赖性抑制同基因或异基因脾细胞对有丝分裂原和异基因抗原诱发的免疫反应性增殖；在mMSC作用下，脾细胞中CD4^＋／CD8^＋比值增高、CD4^＋CD25^＋细胞比例升高，致炎因子分泌降低、抗炎因子分泌升高，并呈mMSC数量依赖性。

Objective To investigate the effect of murine mesenchymal stem cells （mMSC） on immunoproliferative response of spleen cells. Methods Mitogen （ Con A, 20 μg/ml） or irradiated （20 Gy） allogeneic spleen cells （from BALB/c or C57BL/6 mouse depending on the responder cells） were used as stimulators. Proliferations of the responder cells were determined with MTT on day 3 after culture at 37 ℃, 5% CO2 humidified atmosphere. The ratios of CD4^＋ /CD8^＋ and CD4^＋ CD25^＋ cells were analyzed with FACS assay, and the levels of cytokines in supematants with ELISA. Results （1）mMSC inhibited the response of both syngeneic and allogeneic splenic cells to ConA. At the ratio of mMSC to splenic cells being 1 ： 1, the inhibition rate reached 84.21%. With the ratio decreasing, the inhibition rate decreased. （2）mMSC inhibited the response of both syngeneic and allogeneic splenic cells to alloantigen. When the ratio of mMSC to responder cells was 1： 10, the inhibition rate was as high as 88.07%. （3）mMSC could increase the ratio of CD4^＋/ CD8^＋ T cells and the percentage of CD4^＋ CD25 ^＋ cells in splenic cells. These abilities were in a dose-dependent manner and non-MHC antigen restricted. （4）mMSC decreased interleukin（IL） -2, interferon（IFN） - γ, while increased TGF-β1 and IL-4 in the co-culture system. Conclusion mMSC can suppress proliferative response of splenic cells to mitogen and alloantigen, increase the ratio of CD4^＋/CD8^＋ and the proportion of CD4^＋ CD25^＋ in T cells, decrease the secretion of proinflammatory cytokines and increase the anti inflammatory cytokines in a dose-dependent and non-MHC antigen restricted manner.