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目的研究人脐带间充质干细胞(huMSCs)及其诱导分化为男性生殖细胞样细胞后的免疫学特性,为进一步研究huMSCs的移植特性提供实验依据。方法分离培养huMSCs,在男性生殖细胞培养条件下定向诱导其分化为huMSCs源男性生殖细胞样细胞。采用FACScan流式细胞仪检测诱导前后huMSCs表面抗原CD40、CD40L、CD80、CD86。半定量反转录(RT)-PCR检测诱导前后huMSCs的人类白细胞抗原(HLA)-Ⅰ、HLA-DR基因表达水平。采用CCK-8法检测诱导前后的不同数量级huMSCs对混合淋巴细胞反应体系中经植物血凝素(PHA)激活的同种异体外周血淋巴细胞(PBMC)增殖的影响。采用酶联免疫吸附试验检测诱导前后huMSCs对经PHA活化的T细胞分泌干扰素-γ(IFN-γ)的影响。结果 HuMSCs在男性生殖细胞培养条件下能分化为男性生殖细胞样细胞,诱导前后的huMSCs均表达HLA-Ⅰ,不表达CD40、CD40L、CD80、CD86和HLA-DR。huMSCs能抑制经PHA激活的T淋巴细胞增殖,并抑制T淋巴细胞IFN-γ的分泌,但huMSCs源男性生殖细胞样细胞并不能抑制经PHA激活的T淋巴细胞增殖及IFN-γ的分泌。结论 HuMSCs在体外定向诱导为男性生殖细胞样细胞后,诱导后的细胞与huMSCs一样具有低免疫源性,同时huMSCs在体外具有免疫抑制性,但诱导获得的huMSCs源男性生殖细胞样细胞不能抑制T细胞的增殖。
Objective To study the immunological characteristics of human umbilical cord mesenchymal stem cells (hMSCs) and their differentiation into male germ cell-like cells, and to provide experimental evidence for further study on the transplantation characteristics of huMSCs. Methods HuMSCs were isolated and cultured, and were induced to differentiate into huMSCs-derived male germ cell-like cells under male germ cell culture conditions. The surface antigens CD40, CD40L, CD80, CD86 of huMSCs before and after induction were detected by FACScan flow cytometry. Semi-quantitative reverse transcription (RT) -PCR detection of human leukocyte antigen (HLA) -Ⅰ, HLA-DR gene expression levels before and after induction of huMSCs. The CCK-8 method was used to detect the effect of huMSCs of different magnitudes before and after induction on the proliferation of allogeneic peripheral blood lymphocytes (PBMCs) activated by phytohemagglutinin (PHA) in the mixed lymphocyte reaction system. The effect of huMSCs before and after induction on the secretion of interferon-γ (IFN-γ) by PHA-activated T cells was detected by enzyme-linked immunosorbent assay. Results HuMSCs could differentiate into male germ cell-like cells under the conditions of male germ cell culture. Both of the pre-and post-huMSCs expressed HLA-Ⅰ and did not express CD40, CD40L, CD80, CD86 and HLA-DR. huMSCs can inhibit the proliferation of PHA-activated T lymphocytes and inhibit the secretion of IFN-γ by T lymphocytes. However, huMSCs-derived male germ-cells do not inhibit PHA-activated T lymphocyte proliferation and IFN-γ secretion. CONCLUSIONS: HuMSCs were induced to be human germ-like cells in vitro. The induced cells were of low immunogenicity as huMSCs. At the same time, huMSCs were immunosuppressive in vitro. However, the induced male germ-cell-like cells derived from huMSCs could not inhibit T Cell proliferation.