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目的比较免疫性血小板减少症(immune thrombocytopenia,ITP)患儿与正常人骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)及脐带来源间充质干细胞(umbilical cord mesenchymal stem cells,UC-MSCs)的性质及其对正常人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)分泌干扰素-γ(interferon-γ,IFN-γ)、白介素-10(interleukin-10,IL-10)的调节能力。方法用密度梯度离心法体外分离培养16例ITP患儿和8例正常成人BMMSCs至5-6代,用酶消化法从10例健康胎儿脐带组织中分离培养UC-MSCs至5-6代。在培养过程中观察三种来源间充质干细胞(mesenchymal stem cells,MSCs)形态,进行细胞表面分子及成脂成骨分化鉴定并用细胞增殖检测试剂盒(Cell Counting Kit-8,CCK-8)检测三种MSCs的增殖能力。将上述三种MSCs用丝裂霉素(mitomycin C,MMC)处理后与植物血凝素(phytohaemagglutinin,PHA)刺激的PBMC按不同比例(1∶40,3∶40,9∶40)混合培养3 d,收集培养上清液。用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测各组共培养上清液中IL-10和IFN-γ的含量。结果三种MSCs有相似的细胞形态,UC-MSCs增殖速度最快,正常成人BMMSCs次之,ITP患儿BMMSCs最慢;三种MSCs有相似的分化能力。三种来源MSCs与PBMC以不同比例混合培养,随着MSCs比例的增加,PBMC分泌IL-10增加,在ITP患儿BMMSCs组中不同比例之间差异有显著性(P<0.05),而在正常人BMMSCs、UC-MSCs组中不同比例之间差异有非常显著性(P<0.01);随着MSCs比例的增加,PBMC分泌的IFN-γ减少,在ITP患儿及正常人BMMSCs组中不同比例之间差异有显著性(P<0.05),而在UC-MSCs组中不同比例之间差异有非常显著性(P<0.01)。在相同共培养比例条件下,三种MSCs刺激PBMC分泌IL-10和IFN-γ的量差异均无显著性(P>0.05)。结论 ITP患儿BMMSCs较其他两种来源的MSCs增殖速度慢,说明其体外增殖存在缺陷。但ITP患儿BMMSCs与其他两种来源MSCs对PBMC分泌IL-10、IFN-γ的调节具有相同的特点,即促进IL-10分泌,抑制IFN-γ分泌,均呈剂量依赖性,即三种细胞调节PBMC分泌IL-10及IFN-γ能力未见明显差异。
Objective To compare the effects of immunosuppressive thrombocytopenia (ITP) and normal human bone marrow mesenchymal stem cells (BMMSCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) And its ability to regulate the secretion of interferon-γ (IFN-γ), interleukin-10 (IL-10) from peripheral blood mononuclear cells (PBMCs) . Methods 16 cases of ITP children and 8 normal adult BMMSCs were isolated and cultured in vitro by density gradient centrifugation to 5-6 passages. UC-MSCs were isolated and cultured from umbilical cord of 10 healthy fetuses to 5-6 passages by enzymatic digestion method. The morphology of three kinds of mesenchymal stem cells (MSCs) was observed during culture. The cell surface molecules and adipogenic osteogenic differentiation were identified and detected by Cell Counting Kit-8 (CCK-8) Proliferation of three kinds of MSCs. The above three kinds of MSCs were treated with mitomycin C (MMC) and then mixed with phytohaemagglutinin (PHA) -stimulated PBMCs at different ratios (1:40, 3:40, 9:40) d, collect the culture supernatant. The contents of IL-10 and IFN-γ in the co-culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). Results The three MSCs had similar cell morphology, the fastest proliferation rate of UC-MSCs, followed by normal adult BMMSCs, and the slowest of BMMSCs among ITP children. The three MSCs had similar differentiation ability. The MSCs and PBMCs from three sources were mixed and cultured in different ratios. As the proportion of MSCs increased, IL-10 secretion increased in PBMCs, and there was a significant difference (P <0.05) between the different proportions of BMMSCs in ITP patients. There was a significant difference between different proportions of BMMSCs and UC-MSCs (P <0.01). With the increase of the proportion of MSCs, IFN-γ secreted by PBMC decreased. In different proportion of IMS children and normal BMMSCs, (P <0.05). However, there was a significant difference between different proportions in UC-MSCs group (P <0.01). Under the same co-culture ratio, there was no significant difference in the amount of IL-10 and IFN-γ secreted by PBMC between the three MSCs (P> 0.05). Conclusion The proliferation of BMMSCs in ITP children is slower than that of other two kinds of MSCs, indicating that their proliferation in vitro is deficient. However, BMMSCs of ITP and other two MSCs have the same characteristics of IL-10 and IFN-γ secretion in PBMCs, that is, they promote IL-10 secretion and inhibit IFN-γ secretion in a dose-dependent manner There was no significant difference in the ability of cells to regulate the secretion of IL-10 and IFN-γ by PBMCs.