树突状细胞外泌体诱导间充质干细胞向成骨细胞分化

DC-derived Exosomes Induce Osteogenic Differentiation of Mesenchymal Stem Cells

本研究观察人树突状细胞(dendritic cells,DC)来源的外泌体(DC-derived exosome,DCex)对人骨髓间充质干细胞(mesenchymal stem cells,MSC)成骨分化的影响。采用超速离心法从树突状细胞培养上清中获取DCex,应用电子显微镜观察DCex形态并通过流式细胞术检测和鉴定其细胞来源。取第3代MSC,设3组培养诱导体系:①对照组:α-MEM基础培养液组;②实验组:α-MEM基础培养液+DCex(10μg/ml)组;③α-MEM基础培养液+标准诱导剂组。采用荧光实时定量PCR、琼脂糖凝胶电泳检测各组成骨分化转录因子Runx2 mRNA的表达情况,并进行碱性磷酸酶(ALP)活性检测。另外,荧光染料DiI标记DCex,应用共聚焦显微镜观察DCex进入MSC的过程。结果显示,在电子显微镜下DCex呈典型的圆形或椭圆形膜层结构,直径为40-100 nm,并表达DC细胞的标志物CD83,CD86,CD80和HLA-DR。在DCex处理MSC后6 h时,可在共聚焦显微镜下观察到进入到MSC胞质内的DCex,随着作用时间延长,细胞荧光强度增加。按组培养第7天,DCex实验组Runx2表达较对照组增高,差异有统计学显著性(P<0.05);MSC培养第14天,DCex组ALP含量OD值与对应蛋白比为2.22±0.27,显著高于阴性对照组(1.20±0.21)(P<0.01),但低于标准诱导组(3.22±0.24)(P<0.05)。结论:DCex可以促进MSC向成骨细胞分化,其具体机制仍需进一步研究证实。

This study was aimed to investigate the effect of dendritic cell-derived exosome （DCex） on in vitro osteo blast differentiation of human bone marrow mesenchymai stem cells （ hBM-MSC ）. DCex were harvested from the DC culture supernatants by ultracentrifugation. The morphology of DCex was observed by using transmission electron microscopy and the surface marker expression was detected by flow cytometry. MSCs at passage 3 were used in this study. DCex incorporation into MSCs was observed under a confocal microscope. MSCs were either exposed to DCex （ 10 μg/ ml） or the standard osteogeneic induction condition. The cells cultured in complete medium were served as the control. The expression levels of Runt related transcription factor 2 （Runx2） were detected by real-time and standard PCR. The cellular alkaline phosphatase （ALP） activity was also detected. The results showed that the DCex were spherical or oval membrane vesicles with diameters of about 40 - 100 nm under transmission electron microscope. The DCex expressed surface molecules specific for DCs, including CD83, CD86, CDS0, and HLA-DR. After cultured for 7 days, the MSCs treated with DCex highly expressed Runx2 as compared with the control group （ P 〈 0.05 ）. After cultured for 14 days, ALP activity of the DCex-treated MSCs was markedly higher than the control group （ P 〈 0.01 ）, though it was lower than that of MSCs treated with standard inductive agents. It is concluded that DCex can induce MSCs to differentiate into osteoblasts. The detailed investigations are needed to clarify the underlying mechanisms.