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目的:体外研究免疫性血小板减少症(ITP)患者骨髓间质干细胞(MSCs)对CD4(+)CD25(+)Treg细胞的影响。方法:采用Ficoll分离ITP患者及健康对照者骨髓单个核细胞,通过体外培养,扩增出MSCs。通过Ficoll分离法和尼龙棉柱法获取ITP患者外周血T淋巴细胞,ITP患者MSCs经丝裂霉素C处理后按不同数量(1×103、1×104、1×105个细胞/孔)(A1、A2、A3组)接种培养板作为基底层细胞;健康对照者MSCs经丝裂霉素C处理后按不同数量(1×103、1×104、1×105个细胞/孔)(B1、B2、B3组)接种培养板作为基底层细胞;然后分别以2×105个细胞/孔接种体外分离纯化的ITP患者T淋巴细胞,设立空白组C组(ITP患者T淋巴细胞单独培养)。培养第3天各自收集T淋巴细胞,用流式细胞术检测各组CD4(+)CD25(+)Treg细胞比例。结果:在血凝素作用下,实验组及对照组各浓度亚组CD4(+)CD25(+)Treg细胞数量均较空白组明显减少(P<0.05);A2组较B2组CD4(+)CD25(+)Treg细胞数量显著减少(P<0.05),A3组较A2、B3组CD4(+)CD25(+)Treg细胞数量明显减少(P<0.05)。结论:ITP患者可能存在MSCs质或量的改变,导致免疫调节功能异常,对CD4(+)CD25(+)Treg细胞增殖抑制作用明显加强,CD4(+)CD25(+)Treg细胞的不足引起外周免疫耐受减弱,MSCs的免疫调节功能异常可能在ITP发病过程中起重要作用。
Objective: To study the effect of bone marrow mesenchymal stem cells (MSCs) on CD4 (+) CD25 (+) Treg cells in patients with immune thrombocytopenia (ITP) in vitro. METHODS: Bone marrow mononuclear cells from ITP patients and healthy controls were isolated by Ficoll. MSCs were obtained by in vitro culture. Peripheral blood T lymphocytes from patients with ITP were obtained by Ficoll separation method and nylon cotton column method. The MSCs of ITP patients were treated with mitomycin C at different concentrations (1 × 10 3, 1 × 10 4, 1 × 10 5 cells / well) A1, A2, A3) were inoculated with the culture plates as basal cells. The MSCs of healthy controls were treated with mitomycin C in different numbers (1 × 10 3, 1 × 10 4, 1 × 10 5 cells / well) B2, and B3 groups) were inoculated into basal cells. T lymphocytes from ITP patients were inoculated separately with 2 × 105 cells / well in vitro, and group C (blank control group, T lymphocytes cultured alone) was established. T lymphocytes were collected on the third day of culture, and the proportion of CD4 (+) CD25 (+) Treg cells in each group was detected by flow cytometry. Results: Under the action of hemagglutinin, the number of CD4 (+) CD25 (+) Treg cells in experimental group and control group was significantly lower than that in blank group (P <0.05) The number of CD25 (+) Treg cells was significantly decreased (P <0.05). The number of CD4 (+) CD25 (+) Treg cells in A3 group was significantly decreased than that in A2 and B3 groups (P <0.05). CONCLUSION: ITP patients may have changes in the quality or quantity of MSCs, leading to abnormal immunomodulatory function, significantly inhibiting the proliferation of CD4 (+) CD25 (+) Treg cells, and insufficient CD4 (+) CD25 (+ Immune tolerance weakened, abnormal immunoregulatory function of MSCs may play an important role in the pathogenesis of ITP.