间充质干细胞体外对ITP患者T淋巴细胞分泌功能的影响  

Effect of mesenchymal stem cells on secreting function of T lymphocytes from patients with idiopathic thrombocytopenic purpura in vitro

作　　者：马骥[1] 郭成山[1] 赵霞[2] 贺韦东[3] 王迎雪[1] 董巧凤[4] 赵敬杰[5] 

机构地区：[1]山东大学第二医院血液科,山东济南250033 [2]胜利油田中心医院血液科,山东东营257055 [3]山东省血液中心,山东济南250014 [4]菏泽市立医院血液科,山东菏泽274000 [5]山东大学第二医院分子生物学实验室,山东济南250033

出　　处：《中国病理生理杂志》2009年第8期1570-1575,共6页Chinese Journal of Pathophysiology

基　　金：山东省教育厅科技卫生计划资助项目(No.J06L69);山东省医药卫生科技计划资助项目(No.2007HZ046);山东省自然科学基金重点资助项目(No.Z2008C08)

摘　　要：目的:体外观察间充质干细胞(MSCs)对特发性血小板减少性紫癜(ITP)患者T淋巴细胞分泌细胞因子功能的影响。方法:采用Ficoll分离和体外贴壁、传代培养,扩增出骨髓MSCs;通过Ficoll分离法和尼龙棉柱法获取ITP患者外周血T淋巴细胞。以经丝裂霉素(MMC)处理后不同数量(2×103、1×104、5×104cells/well)的MSCs作为基底层细胞,接种体外分离纯化的异体ITP患者T淋巴细胞,分别于2d、4d、6d后各自收集培养上清,用酶联免疫吸附试验(ELISA)法动态测定T淋巴细胞分泌白细胞介素2(IL-2)、干扰素-γ(IFN-γ)、白细胞介素4(IL-4)、白细胞介素10(IL-10)水平的变化。结果:ITP患者T淋巴细胞分泌细胞因子IL-2、IFN-γ较正常人高(P<0.05),IL-4、IL-10较正常人低(P<0.05)。MSCs可显著抑制ITP患者或正常对照组T淋巴细胞分泌IL-2、IFN-γ(P<0.05),且随MSCs数量的增加,抑制增强(P<0.05),共培养4d、6d时作用明显强于2d时(P<0.05);MSCs可促进ITP患者T淋巴细胞分泌IL-4、IL-10(P<0.05),且随MSCs量的增加,促进作用增强(P<0.05),对IL-10的作用随时间延长而增强(P<0.05),但对IL-4的作用在培养第2d、4d、6d时无显著差异(P>0.05);在正常对照组,当MSCs数量>1×104可以促进T淋巴细胞分泌IL-4与IL-10(P<0.05),且随MSCs数量的增加,作用增强(P<0.05),共培养4d、6d时作用明显强于2d时(P<0.05)。结论:MSCs能够在体外调节ITP患者辅助性T细胞1(Thl)和辅助性T细胞2(Th2)反应平衡,可使ITP患者Th1极化状态部分改善。AIM： To analyze the effect of mesenchymal stem ceils （MSCs） on secreting cytokines by T lymphocytes from patients with idiopathic thrombocytopenic purpura （ITP） in vitro. METHODS： Human bone marrow -derived MSCs were isolated by Ficoll Hypaque ： nd cultured for proliferating to passage cells. Allogeneic T lymphocytes of ITP were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column. Then the stromal feeder layers of different numbers （2 × 10^3, 1 × 10^4, 5 × 10^4 per well） of MSCs treated with mitomycin were co - cultured with above - mentioned T lymphocytes. The supernatant were respectively collected on day 2, 4 and 6 after co - culture, then the levels of IL - 2, IFN-γ, IL-4, IL- 10 secreted by T lymphocytes were measured by enzyme linked immunosorbent assay （ELISA） drnamically. RESULTS ： The levels of IL - 2 and IFN -γ/secreted by T cells from ITP were higher than those from normal control （P 〈0. 05, respectively）. Inversely, IL-4 and IL- 10 were lower than those in normal control （P 〈0. 05, respectively）. After co - cultured with T lymphocytes, MSCs significantly inhibited the cytokine levels of IL -2 and IFN - γ secreted by T lymphocytes from ITP or health adults （ P 〈 0. 05, respectively） in a dose dependent manner （P 〈 0.05, respectively）, and the effect was more obvious when co- cultured for 4 days or 6 days than that for 2 days （P 〈 0. 05, respectively）. However, MSCs significantly promoted the releases of IL -4 and IL - 10 by T lymphocytes from ITP patients （P 〈0. 05, respectively） in a dose dependent manner （P 〈0. 05, respectively）, and the effect on IL - 10 was in a time dependent way （P 〈0. 05）, while the effect on IL-4 had no obvious difference among 2 d, 4 d and 6 d（P 〉0. 05）. As for health control group, when cell numbers exceeded above 1 ×10^4, MSCs obviously promoted IL -4 and IL - 10 levels secreted by T lymphocytes （ P 〈 0. 05 ） in a dose dependent manner （P 〈 0. 05 ）, and both

关 键 词：间充质干细胞 特发性血小板减少性紫癜 细胞因子类 T淋巴细胞 

分 类 号：R392.11[医药卫生—免疫学]