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目的观察人羊膜间充质干细胞(hAMSC)对体外培养的淋巴细胞功能的影响。方法通过酶消化法分离培养hAMSC,采用荧光团标记的小鼠抗人单克隆抗体结合流式细胞术鉴定细胞表面抗原;免疫荧光染色检测培养细胞波形蛋白(vimentin)和阶段特异表达抗原4(SSEA-4)的表达;分离培养hAMSC和外周血单个核细胞(PBMC),将刀豆蛋白(ConA)刺激的淋巴细胞与1×104、5×104、1×105个hAMSC进行共培养。CCK-8法测定淋巴细胞增殖,ELISA测定细胞上清液中IFN-γ的水平。结果5μg/mLConA能够引起淋巴细胞增殖;共培养条件下,hAMSC能够抑制ConA引起的淋巴细胞增殖,且随着hAMSC的数量增加,抑制效果更明显。培养72h后,CCK-8法结果表明,单纯ConA刺激后淋巴细胞数显著高于共培养细胞。选择抑制效果最佳的组别1×106个淋巴细胞与1×105个hAMSC共培养,ELISA测定1×105个hAMSC对淋巴细胞抑制72h后,上清液中IFN-γ分泌,共培养细胞上清液中IFN-γ水平显著低于单纯ConA刺激细胞。结论hAMSC能在体外抑制ConA引起的淋巴细胞增殖并减少IFN-γ分泌。
Objective To observe the effect of human amniotic mesenchymal stem cells (hAMSC) on lymphocyte function in vitro. Methods The hAMSCs were isolated and cultured by enzymatic digestion, and the cell surface antigens were detected by fluorophore-labeled mouse anti-human monoclonal antibodies combined with flow cytometry. The expressions of vimentin and phase-specific antigen 4 (SSEA -4). HAMSC and peripheral blood mononuclear cells (PBMCs) were isolated and cultured. ConA-stimulated lymphocytes were co-cultured with 1 × 104, 5 × 104 and 1 × 105 hAMSCs. Lymphocyte proliferation was measured by CCK-8 assay and IFN-γ level in supernatant was measured by ELISA. Results 5μg / ml LConA can induce lymphocyte proliferation. Under co-culture conditions, hAMSC can inhibit the proliferation of lymphocytes induced by ConA, and with the increase of hAMSC, the inhibitory effect is more obvious. After cultured for 72h, the results of CCK-8 assay showed that the number of lymphocytes after ConA stimulation was significantly higher than that of cocultured cells. 1 × 106 lymphocytes and 1 × 105 hAMSCs were selected to co-culture with the best inhibitory effect. The inhibitory effect of 1 × 105 hAMSC on lymphocytes was measured by ELISA after 72 h. IFN-γ secretion in the supernatant, co-cultured cells Serum IFN-γ levels were significantly lower than the ConA-stimulated cells alone. Conclusion hAMSC can inhibit ConA-induced lymphocyte proliferation and decrease IFN-γ secretion in vitro.