摘要
[目的]观察并分析健康供者的骨髓间充质干细胞(mesenchymal stem cells,MSCs)对免疫性血小板减少症(immune thrombocytopenia,ITP)患者外周血T淋巴细胞亚群及T淋巴细胞因子分泌水平的影响,探讨MSCs对ITP患者T淋巴细胞分化及分泌功能的影响。[方法]Ficoll液密度离心法联合尼龙毛柱法分离健康供者/ITP患者外周血T淋巴细胞;离心法分离外周血血清;Ficoll液密度离心法分离健康供者骨髓液单个核细胞,贴壁法分离培养出MSCs;以健康供者/ITP患者的T淋巴细胞和自身血清为基础,加入不同浓度的MSCs,设为试验组(T淋巴细胞和MSCs比值:1∶0.01、1∶0.02、1∶0.08)、空白组(单纯健康供者/ITP患者T淋巴细胞),培养72h;流式细胞仪检测MSCs纯度及共培养体系中的T淋巴细胞亚群比例;酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测培养上清液中淋巴细胞因子白介素-2(interleukin-2,IL-2)、白介素-10(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、干扰素-γ(interferon-γ,IFN-γ)、IL-4水平变化。[结果]加入MSCs共培养后,健康供者和ITP患者各组内CD4+T淋巴细胞变化均无统计学意义,健康供者组内调节性T淋巴细胞(regulatory T cell,Treg)比值随MSCs浓度的增加而升高(P=0.012),而ITP患者组内Treg比值差异无统计学意义;未加MSCs的情况下,ITP患者空白组的IL-2、TNF-α及IFN-γ水平高于健康供者空白组,差异有统计学意义(P<0.01),IL-10水平低于健康供者空白组,差异有统计学意义(P=0.002);加入MSCs(1:0.08比例)共培养后,ITP患者组的CD8+T淋巴细胞低于空白组,差异有统计学意义(P=0.003),IL-2、TNF-α、IFN-γ水平低于空白组,差异有统计学意义(P<0.01),IL-10水平显著上升(P<0.01)。[结论]与健康供者相比,ITP患者IL-2、TNF-α及IFN-γ的分泌增多,IL-10分泌减少。MSCs在体外可下调ITP患者CD8+T淋巴细胞增殖,抑制ITP患者Th1类细胞因子的表达。
[Objective]To study the T lymphocytes subsets and their cytokines in patients with ITP after co-cultured with human bone marrow-derived MSCs from healthy donors in vitro. To explore the MSCs＇ immunomodulatory effect on T lymphocytes in patients with ITP. [Methods]T cells were obtained from peripheral blood of both healthy donors and ITP patients by Ficoll Hypaque and nylon cotton column. Serum was separated by centrifugalization.Human bone marrow-derived MSCs were isolated from healthy donors＇ bone marrow mononuclear cells by Ficoll Hypaque. The co-culture system was mainly comprised of MSCs, T lymphocytes and self-serum. After 72 hours co-culture, T lymphocyte subsets and cytokines in the co-culture system were measured by flow cytometry and ELISA, respectively. [Results] After co-cultured with MSCs,CD4＋T cells proportions didn＇t differ inter-ITP groups, so did that inter-healthy donor group（for healthy donor group, P=0.775;for ITP group, P=0.630）. Treg ratio inter-healthy donor groups increased in an MSCs concentration-dependent way（P=0.012）, whereas no difference was found in that inter-ITP groups（P=0.067）. In comparison to that of healthy donor blank group, the secretion of IL-2, TNF-α and IFN-γ in ITP blank group increased（P0.01）, whereas decreased the secretion of IL-10（P=0.002）. After co-cultured with MSCs（ratio of 1：0.08）, the proportion of CD8＋T cells in ITP group decreased（P=0.003）. And cytokine level in ITP group changed as well, manifesting as decreased level of IL-2, TNF-α, IFN-γ, and increased level of IL-10（P0.05）. [Conclusions]The secretion of IL-2, TNF-α and IFN-γ increased in patients with ITP, whereas decreased the IL-10. Bone marrow-derived MSCs in vitro down-regulated the expression of CD8＋T cells of ITP patients, as well as the secretion of Th1-associated cytokines.
出处
《浙江中医药大学学报》
CAS
2016年第4期245-252,共8页
Journal of Zhejiang Chinese Medical University
基金
"十一五"国家科技支撑计划(2007BAI20B064)
国家自然科学基金(81373634)
浙江省医药卫生科研项目(2010KYB077)
浙江省中医药优秀青年人才基金计划(2015ZQ012)~~
