冷冻前后人脐带间充质干细胞免疫抑制能力的比较

Comparison of immunosuppressive capability of human umbilical cord mesenchymal stem cells before and after cryopreservation

目的探讨冷冻对人脐带间充质干细胞(h UC-MSC)免疫调节功能的影响。方法对足月剖宫产新鲜脐带采用组织贴块法分离h UC-MSC,贴壁培养,传代纯化冷冻后,在体外将γ-干扰素(IFN-γ)刺激后新鲜和冻融h UC-MSC与混合淋巴细胞共培养,实验检测激活的新鲜和冻融h UC-MSC对混合淋巴细胞增殖抑制的变化。用实时荧光定量聚合酶链反应(PCR)实验和酶联免疫吸附分析(ELISA)实验检测激活后新鲜和冻融h UC-MSC基因吲哚胺-2,3-双加氧酶(IDO)、转化生长因子β(TGF-β)、环氧合酶2(COX-2)m RNA和前列腺素E2(PGE2)的表达变化情况。结果 IFN-γ可以增强新鲜和冻融h UC-MSC对活化的人外周血单个核细胞(h PBMC)的增殖抑制能力,但冻融h UC-MSC对活化的h PBMC的增殖抑制能力明显弱于新鲜h UC-MSC增殖抑制能力(P<0.05)。IFN-γ刺激24 h后,上调新鲜和冻融h UC-MSC表达免疫抑制基因IDO m RNA和TGF-βm RNA,但冻融h UC-MSC表达免疫抑制基因IDO m RNA和TGF-βm RNA低于新鲜h UC-MSC(P<0.05)。IFN-γ刺激24 h后,h UC-MSC分泌PGE2具有降低趋势,并且冻融h UC-MSC分泌PGE2水平明显少于新鲜h UC-MSC的分泌水平(P<0.05)。IFN-γ刺激24 h后冻融h UC-MSC比新鲜h UC-MSC表达环氧合酶2(COX-2)明显降低(P<0.05),并且冻融h UC-MSC比新鲜h UC-MSC表达COX-2明显降低(P<0.05)。结论冷冻保存h UC-MSC免疫抑制潜能被保留,这些细胞保持其抑制混合淋巴细胞增殖的能力,但其激活的能力下降。

Objective To explore the effect of cryopreservation on immunosuppressive capability of human umbilical cord mesenchymal stem cells（hUC-MSC）. Methods hUC-MSC were obtained by tissue isolation, and the cells were purified by adherent culture and cryopreservation. The proliferation of human peripheral blood mononuclear cells（hPBMC） was evaluated after co-culture with hUC-MSC and interferon-y（IFN-3,） treatment. Real-time polymerase chain reaetion（PCR） and enzyme- linked immunosorbent assay（ELISA） were carried out to test the expression changes of fresh and freeze-thaw activated hUC- MSC gene indoleamine-2, 3-dioxygenase（IDO）, transforming growth factor beta（TGF-β） mRNA and prostaglandin E2（PGE2 ）. Results IFN-y could enhance the suppression of mitogen stimulated proliferation of activated hPBMC by fresh and frozen thawed hUC-MSC, while the proliferation inhibition ability of thawed hUC-MSC on activated hPBMC was significantly weaker than that of fresh hUC-MSC. After stimulated by IFN-y, the immunomodulatory property of hUC-MSC on the proliferation of hPBMC was enhanced（P 〈 0.05）. After IFN-y stimulation for 24 hours, compared with control group, IDO and TGF-[3 expression level increased significantly（P 〈 0.05）, the production of PGE2 secreted by thawed hUC-MSC decreased significantly compared with that of fresh（P 〈 0.05）, and the mRNA expression level of COX-2 also decreased signifieant（P 〈 0.05）. Conclusion It is demonstrated that the immunosuppressive potential of hUC-MSC which inhibit the proliferation of mixed lymphocytes are pre- served after freezing, while the activation ability are weakened.