免疫性血小板减少症患儿骨髓间充质干细胞与其他来源的间充质干细胞免疫调节作用的比较研究

The different immunomodulation of the mesenchymal stem cells from ITP patients bone marrow and other origins

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 cell morphology, growth rate, differentiation capacity and the ability of regulating peripheral blood mononuclear cell （PBMC） to secrete IL-10 and IFN-γ/by bone marrow mesenchymal stem cells （BMMSCs） which were isolated from immune thrombocytopenia （ITP） patients, healthy adults and umbilical cord mesenchymal stem cells （UC-MSCs）. Methods The 5-6 generations of BMMSCs were isolated from ITP patients （n = 16） and healthy adults （n = 8） using density gradient centrifugation. The 5-6 generations of UC-MSCs were isolated from healthy fetuses＇ umbilical cord using enzyme digestion. In the process of culture, the morphology of three kinds of MSCs were observed ; the immunophenotype, adipogenic and osteogenic differentiation potential of them were identified; the reproductive activity of them was assessed by cell counting kit-8 （CCK-8）. Besides, three kinds of MSCs were inactivated by mitomyein C （MMC） and co-cultured with phytohaemagglutinin （PHA）-activated PBMC with the ratio of 1：40, 3：40 and 9.40 respectively. Three days later, the culture supernatant was collected and the eytokines IL-10 and IFN-~ were measured by ELISA. Results Three kinds of MSCs had similar cell morphology, immunophenotype and differentiation potential. UC-MSCs showed the fastest growth rate ; BMMSCs of ITP patients was the slowest one and the BMMSCs of healthy adults was between them. Three kinds of MSCs were co-cultured with different concentrations of PBMC. The results showed that the more propotions of MSCs, the more increase of IL-10 （in BMMSCs of ITP patients group, P 〈 0. 05 ; in BMMSCs of healthy adults and UC-MSCs groups, P 〈0. 01 ） ;conversely, the more propotions of MSCs, the more decrease of IFN-γ （ in BMMSCs of ITP patients and healthy adults groups, P 〈 0. 05 ; in UC-MSCs group, P 〈 0. 01 ）. There was no significant difference among three groups when co-cultured in the same conditions （P 〉 0. 05 ）. Conclusions Three kinds of MSCs had the same characteristics in regulating PBMC to secrete IL-10 and IFN-γ： promoting the secretion of IL-10 and inhibiting the secretion of IFN-γ levels by PBMC in a dose-dependent manner. There was no significant difference among MSCs from different origins in regulating PBMC to secrete IL-10 and IFN-γ.