骨髓间充质干细胞重塑枯否细胞表型诱导免疫耐受

Interaction of bone marrow stromal cells with Kupffer cells induces immune tolerance

目的 建立骨髓间充质干细胞（BMSCs）与枯否细胞（KCs）的体外共培养体系,并观察BMSCs对KCs表型的影响,为BMSCs在肝脏移植中的应用提供依据.方法 建立KCs与BMSCs（1：1）直接接触共培养体系作为实验组,单独培养KCs为对照组.两组细胞均加入内毒素（LPS,1 mg/L）,培养24h后,应用实时定量聚合酶链反应（Real-time PCR）法及Western blot法检测白细胞介素（IL）-10基因及蛋白的表达、酶联免疫吸附试验（ELISA）法检测细胞IL-10、前列腺素E2（PGE2）、IL-6分泌量、流式细胞仪检测人类主要组织相容性复合体（MHC）Ⅱ、CD40、CD80、CD86表达.结果 Real-time PCR及Western blot检测结果显示,与对照组比较,共培养体系能明显增加IL-10基因和蛋白质的表达（P＜0.05）.LPS刺激后,共培养组IL-10分泌量（2 307.44±308.97） ng/L与PGE2分泌量（424.48 ±47.86） ng/L均比对照组高[IL-10：（1 000.12±49.06） ng/L;PGE2：（244.48 ±28.15） ng/L,P ＜0.01]；IL-6分泌量低于对照组[BMSCs＋ KCs：（78.14±10.58） ng/L;KCs：（283.18 ±20.50）ng/L,P＜0.01].流式细胞细胞仪检测显示,LPS刺激24h后,与对照组高表达MHCⅡ（99.80％）、CD40（99.26％）、CD80（99.93％）及CD86（99.92％）比较,共培养组MHCⅡ （95.18％）、CD40（89.92％）、CD80（99.89％）、CD86（94.75％）表达下调.结论 BMSCs能干扰KCs功能表达,重塑KCs表型,诱导KCs向M2型巨噬细胞转变,产生抑制免疫应答效应.

Objective To investigate the phenomenon of bone marrow stromal cells （BMSCs） regulating the function of Kupffer cells （KCs） under direct contact co-culture in vitro.Methods In co-culture experiments,pure rat BMSCs were directly added to the KCs plates with the ratio of 1∶ 1.KCs were separately cultured in a well as control group.After stimulation with lipopolysaccharide （LPS,1 mg/L） for 24 h,the gene and protein expression of interleukin （IL）-10 was detected by using real-time quantitative polymerase chain reaction （Real-time PCR） and Western blotting,respectively.The abundance of IL-10,prostaglandin E2 （PGE2） and IL-6 was detected by using enzyme linked immunosorbent assay （ELISA） kits.Flow cytometry was used to detect the expression of major histocompatibility complex （MHC） Ⅱ,CD40,CD80 and CD86.Results As compared with control group,the gene and protein expression of IL-10 in co-culture system was significantly increased （P 〈 0.05）.After co-culture with BMSCs,the levels of IL-10 [KCs：（1000.12±49.06） ng/L; KCs ＋ BMSCs：（2307.44 ±308.97） ng/L] and PGE2[KCs：（244.48 ±28.15） ng/L; KCs ＋ BMSCs：（424.48 ± 47.86） ng/L] were significantly increased （P 〈 0.01）,while the abundance of IL-6 [KCs：（283.18 ± 20.50） ng/L; KCs ＋ BMSCs：（78.14 ± 10.58） ng/L] was significantly down-regulated （P 〈 0.01）.In co-culture system,the expression of MHC Ⅱ,CD40,CD80 and CD86 was lower than in control group.Conclusion BMSCs may reprogram the expression patterns of KCs,regulate the function of KCs,and then induce immune tolerance.