骨髓间充质干细胞对外周血T淋巴细胞亚群影响的体外实验研究

Study on the ex vivo effect of mesenchymal stem cells on peripheral subsets of T lymphocyte

目的:探讨骨髓间充质干细胞(MSC)在体外对T淋巴细胞亚群及其分泌的细胞内因子的影响。方法:通过Ficoll-Hypaque梯度密度离心法分离出正常人骨髓单个核细胞,体外培养扩增MSC,获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养(MLC)体系中,在第1,3,5天,采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况,再用流式细胞术分析各组T细胞亚群及其内因子白介素(IL)-4和干扰素(IFN)-γ的分泌变化情况。结果:加入了MSC的MLC体系中,MSC对T淋巴细胞的增殖抑制具有剂量依赖性,且随着时间延长,抑制程度增强;其中,CD4+T细胞亚群受抑不如CD8+T细胞亚群显著;而根据T细胞内因子的变化,Th1和Tc1的比率较对照组有显著降低,而Th2和Tc2的比率却有轻度上升。结论:MSC在体外能够明显抑制T淋巴细胞的增殖,尤其是CD8+T细胞(CTL)。除此之外,它还能降低MLC体系中的Th1和Tc1,升高Th2和Tc2。所以在临床上可能有减轻移植后急性移植物抗宿主病(aGVHD)的发生,而保留移植物抗白血病(GVL)的潜力。

Objective.To investigate the effect of mesenchymal stem cell （MSC） on cytokines and subsets of T lymphocyte in vitro. Method： Normal human bone marrow mononuclear cells were isolated by Ficoll-Hypaque density gradient, ex vivo MSCs were culture-expanded, obtained after the third passage. The MSC were added to the two-way mixed lymphocyte culture （MLC） according to different proportion. On day 1, 3 and 5, the proliferation of T lymphocytes of each group of MLC-MSC and ctrl-MLC was measure with MTT coloremetry. The subsets and Cytokines of T lymphocytes were analyzed by flow cytomety before and on 3th day after co-culture of MLC with MSC. Result. MLC added MSC induce dose-dependent inhibition of T lymphocytes proliferation, and more effectively in company with co-culture time; CD4^＋ T cell subsets was not suppressed as obviously as CD8^＋ T cell subsets; Th1 and Tc1 decrease significantly than control cultures without MSC. But Th2 and Tc2 slightly increased in all experiments. Conclusion： MSC can significantly suppress T-lymphocyte proliferation, especially CD8^＋ T cell subsets, Otherwise it can decrease activated Th1 and Tc1, increase Th2 and Tc2. MSC have potential of alleviating acute graft-versus-host disease（aGVHD） and maintaining graft versus leukemia （GVL）.