骨髓间充质干细胞通过JAK／STAT3信号通路抑制树突状细胞成熟

骨髓间充质干细胞（bone marrow mesenchymal stem cells，bMSCs）具有自我更新、支持造血、多向分化和低免疫原性等特点，在调控树突状细胞（dendritic cells，DCs）成熟的过程中发挥重要作用。为了探讨bMSCs调控DCs成熟的机制，本研究通过分离培养正常捐献者bMSCs，并分离获取外周静脉血单个核细胞，诱导未成熟的树突状细胞（immature dendritic cells，imDCs）和成熟的树突状细胞（mature dendritic cells，mDCs）生成。根据Genebank中人STAT3全长基因序列，设计针对STAT3的siRNA。根据培养条件不同设计实验分组：正常bMSCs与imDCs共培养（阴性对照组），转染siRNA的bMSCs与imDCs共培养（siRNA组）、加入JAK/STAT通路抑制剂AG490的bMSCs与imDCs共培养（AG490组）、加入TNF-α诱导的mDCs（阳性对照组）共4组，共培养72 h，流式细胞术分析DCs表型变化，ELISA检测培养液上清中IL-12水平变化。结果显示，阴性对照组不表达CD40、CD80、CD83、CD86和HLA DR标志树突细胞成熟的分子，而表达CD11b，其表型与imDCs一致；而siRNA组和AG490组的DCs表达CD40、CD80、CD83、CD86和HLA-DR等标志分子，而不表达CD11b，其表型与TNF-α诱导成熟的mDCs表型一致；siRNA组、AG490组和阳性对照组的IL-12水平较阴性对照组的IL-12水平显著升高（P＜0.05），但siRNA组、AG490组和阳性对照组之间无明显差异（P＞0.05）。以上结果表明，通过siRNA和抑制剂AG490阻断bMSCs中JAK/STAT3通路促进了imDCs的成熟，提示bMSCs通过JAK/STAT3通路参与调控imDCs成熟。

Bone Marrow Mesenchymal Stem Cells Inhibit Dendritic Cell Maturation via the JAK/STAT3 Signaling Pathway

Mesenchymal stem cells (MSCs) are capable of self-renewal, multi-directional differentiation, supporting hematopoiesis and immunoregulatory function, which plays important roles in the process of dendritic cell (DC) maturation. In order to investigate the mechanism of bone MSCs (bMSCs) in regulating DC maturation by the JAK/STAT3 pathway, bMSCs derived from bone marrows of healthy donors were isolated, cultured and identified. The mononuclear cells were obtained form peripheral venous blood, and then immature DCs (imDCs) and mature DCs (mDCs) were induced via TNF-α and identified by flow cytometry (FACS). The siRNA targeting STAT3 was designed according to the sequence of GenBank. Then we designed four experimental groups based on the different culturing conditions: normal bMSCs and imDCs (Negative control group), bMSCs co-cultured with imDCs transfected with siRNA targeting STAT3 (siRNA group), bMSCs co-cultured with imDCs added with the AG490 inhibitor (AG490 group) and TNF-α inducible mDCs (Positive control group). After 72 hours of co-culturing, DCs were analyzed by flow cytometry, and interleukin-12 (IL-12) levels in the supernatant were detected by the Enzyme-linked immunosorbent assay test (ELISA). The results showed that the Negative control group did not express CD40, CD80, CD83, CD86 or HLA-DR, but did express CD11b. This pattern is consistent with imDCs. The siRNA and AG490 groups expressed DCs, CD83, CD40, CD80 CD86 and HLA-DR molecular markers representing mature DCs, but did not express CD11b. This pattern is consistent mDCs. IL-12 levels in the siRNA, AG490 and Positive control groups were significantly increased compared with the Negative control group (all P< 0.05), but the levels were not significant different (all P > 0.05) among the siRNA, AG490 and Positive control groups. These results suggest that blocking the JAK/STAT3 pathway in bMSCs through siRNA and the AG490 inhibitor promotes the maturation of imDCs, suggesting that bMSCs regulate imDC maturation through the JAK/STAT3 pathway.