人牙髓间充质干细胞外泌体对小鼠树突状细胞成熟和功能的抑制作用

Inhibitory effects of exosomes derived from human dental pulp mesenchymal stem cells on the maturation and function of dendritic cells in mice

目的探讨人牙髓间充质干细胞来源外泌体(hDPSC-exosomes)对脂多糖(lipopolysaccharide,LPS)刺激的小鼠树突状细胞(dendritic cells,DC)成熟和功能的影响,评价其对免疫系统的调节作用。方法原代培养成人恒牙牙髓间充质干细胞,并提取细胞培养基中的外泌体,透射电镜观察其形态及粒径,Western blot检测其表面CD9、CD63的表达。分别用PBS、LPS、LPS+hDPSC-exosomes刺激小鼠骨髓来源的树突状细胞(DC2.4),24h后采用流式细胞仪检测其表面共刺激分子的表达,ELISA检测细胞因子的分泌水平,RT-PCR方法检测其TLR2、TLR4和NF-κB的mRNA表达水平,混合淋巴细胞反应(MLR)检测hDPSC-exosomes对DC功能的影响。结果成功分离成人恒牙牙髓间充质干细胞,其表面高表达CD73、CD90,低表达CD45,hDPSC-exosomes电镜下呈圆形或椭圆微囊小体,直径50~80nm,其表面标志CD9、CD63表达阳性。hDPSC-exosomes能显著降低LPS刺激后DC表面共刺激分子CD11c和CD86的表达;增加TGF-β降低IL-4的表达水平;同时,hDPSC-exosomes能显著抑制LPS诱导的DC促淋巴细胞增殖的能力;与空白对照组相比,hDPSC-exosomes能促进DC表面TLR2、TLR4mRNA的表达,上调NF-κBmRNA的表达。结论本实验结果表明,hDPSC-exosomes能抑制小鼠DC的活化和功能成熟,通过TLR-NF-κB信号通路,促使其向耐受型DC方向发展,诱导免疫耐受调节免疫平衡。

Objective To investigate the effects of exosomes derived from human dental pulp mesenchymal stem cells (hDPSC-exosomes) on the maturation and function of dendritic cells (DC) stimulated by lipopolysaccharide (LPS), and to evaluate their regulatory effects on the immune system. Methods Adult permanent teeth-derived dental pulp mesenchymal stem cells were cultured in vitro to extract exosomes in the cell culture medium. The morphology and sizes of the exosomes were observed under transmission electron microscopy. Expression of CD9 and CD63 on the surface of the exosomes was detected by Western blot. PBS, LPS and LPS+ hDPSC-exosomes were respectively used to stimulate mouse bone marrow-derived dendritic cells (DC2.4) for 24 h. A blank control group was set up accordingly. Expression of co-stimulatory molecules and cytokine secretion were detected by flow cytometry and ELISA, respectively. Expression of TLR2, TLR4 and NF-κB at mRNA level was detected by RT-PCR. Changes in the functions of DC were evaluated by mixed lymphocyte reaction (MLR). Results Adult permanent teeth-derived dental pulp mesenchymal stem cells were successfully isolated. Up-regulated CD73 and CD90, and down-regulated CD45 were detected on the surface of the cells. Under electron microscopy (SEM), hDPSC-exosomes showed round or oval microcapsule bodies about 50-80 nm in diameter with positive surface markers of CD9 and CD63. hDPSCs-exosomes could significantly reduce the LPS-induced expression of co-stimulatory molecules CD11c and CD86 on DC surface. Moreover, hDPSCs-exosomes increased TGF-β expression and decreased IL-4. They could also significantly inhibit the proliferation of splenic lymphocytes that was induced by DC after LPS stimulation. Compared with the blank control group, hDPSC-exosomes could promote the expression of TLR2 and TLR4 on DC surface and up-regulate the expression of NF-κB. Conclusions This study showed that hDPSC-exosomes could inhibit the activation and functional maturation of DC, promote the development towards tolerant DC through TLR-NF-κB signaling pathway, and induce immune tolerance to regulate immune balance.