间充质干细胞体外对ITP患者CD4^+CD25^+T细胞影响

Effect of mesenchymal stem cells on CD4^+CD25^+T cell from patients with immune thrombocytopenia in vitro

目的:体外观察间充质干细胞(MSCs)对免疫性血小板减少症(ITP)患者CD4+CD25+T细胞比例的影响。方法:采用Ficoll分离骨髓单个核细胞,通过体外培养,扩增出MSCs,通过Ficoll分离法和尼龙棉柱法获取正常人及ITP患者外周血T淋巴细胞,并应用流式细胞术检测T细胞中CD4+CD25+T细胞比例;MSCs经丝裂霉素MMC处理后按不同数量(2×103、1×104、5×104个细胞/孔)接种培养板作为基底层细胞,然后分别接种体外分离纯化的异体ITP及正常人T淋巴细胞,于2、4、6天后各自收集T淋巴细胞及培养上清,用流式细胞术检测接种于骨髓MSCs的ITP患者CD4+CD25+T细胞比例。结果:ITP患者外周血CD4+CD25+T细胞数量及CD4+CD25+/CD4+比值均明显低于正常对照组(P<0.05);在PHA作用下,数量>1×104的骨髓MSCs与T淋巴细胞共培养4天后,与正常对照组相比,MSCs可显著上调ITP患者及正常人T淋巴细胞中CD4+CD25+T淋巴细胞比例及CD4+CD25+/CD4+比值(P<0.05),且随MSCs量的增加,作用增强(P<0.05)。体外骨髓MSCs对ITP患者CD4+CD25+T淋巴细胞具有上调作用,以上这种机制可使ITP患者的细胞因子及CD4+CD25+T淋巴细胞逐渐接近于正常人但仍达不到正常人水平(P<0.05)。结论:MSCs在体外可能通过上调CD4+CD25+调节性T细胞,进而诱导ITP患者免疫耐受形成。

Objective：To analyze the effect of human bone marrow-derived mesenchymal stem cells（MSCs） on ratio of CD4^＋CD25^＋ T cells.Methods： Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells.Allogeneic T lymphocytes of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column,and their ratio of the CD4^＋CD25^＋ T cells was detected by flow cytometry.Then the stromal feeder layers of different amount（2×103,1×104,5×104） per well of MSCs dealed with mitomycin were cocultured with above-mentioned T lymphocytes,and T lymphocytes and supernatant were collected on day 2,4 and 6 after cocultivation,respectively,then the ratio of CD4^＋CD25^＋ T cells in every experimental group was detected by flow cytometry.Results： The amount of CD4^＋CD25^＋ T cells and the ratio of CD4^＋CD25^＋/CD4＋ in the peripheral blood of ITP patients was obviously lower than normal control（P0.05,respectively）.While the amount of MSCs exceeded 1×104 and cocultured with T lymphocytes with PHA-activating for 4 days,MSCs could obviously up-regulate the ratio of CD4^＋CD25^＋ T cells and significantly increase CD4^＋CD25^＋/ CD4＋ in the peripheral blood of ITP patients and health adults compared to control（P0.05,respectively） in a dose dependent way（P0.05,respectively）.As for ITP patients,whose T lymphocytes and CD4^＋CD25^＋T cells regulated by MSCs,and even the amount of CD4^＋CD25^＋ T cells gradually tended to normal level but not reached that yet in vitro（P0.05,respectively）.Conclusion： ITP is a kind of Th1-dominant autoimmune diseases having abnormal T cytokines profile,and the lower quantity of CD4^＋CD25^＋ T cells also suggests that CD4＋ CD25＋ T cells could play an important role in the course of ITP.