调节性T细胞外泌体对牙周膜干细胞成骨分化能力的影响

The effect of T-regulatory-cell-derived exosomes on the osteogenic differentiation of periodontal ligament stem cells

目的研究调节性T细胞(regulatory T cells,Tregs)外泌体(exosomes)对牙周膜干细胞(PDLSCs)骨分化能力的影响。方法采用酶消化法分离及培养PDLSCs,通过流式细胞仪分选人外周血中Treg细胞,使用超速离心法提取Treg细胞外泌体,并进行蛋白鉴定,电镜以及粒径分析。在PDLSCs中加入不同浓度的treg外泌体,分别是1×10^(8),1×10^(10)particles/ml,并进行共培养。使用Real-time RT-PCR和Western Blot技术来检测7 d和14 d的成骨诱导后PDLSCs中Runx2和Osterix mRNA和蛋白的表达。使用茜素红染色技术进行评估,以了解成骨诱导21 d后钙结节形成的情况。通过Western blot检测treg细胞外泌体作用前后PDLSCs内Hes-1蛋白表达情况。结果Treg细胞外泌体的形态为直径约139 nm的圆形或椭圆形囊泡。蛋白检测结果显示提取的treg外泌体中高表达CD63、TSG101、CD9和CD81。Treg细胞外泌体促进了PDLSCs的Runx2和Osterix mRNA及蛋白的表达,茜素红染色显示外泌体组的矿化结节较对照组明显增加。与对照组相比,Treg细胞外泌体组PDLSCs Hes-1蛋白表达量升高。结论Treg细胞外泌体可以促进PDLSCs成骨分化能力,推测可能与其激活Notch信号通路有关。

Objective To investigate the effect of exosomes derived from regulatory T cell(treg)on the osteogenic differentiation of periodontal ligament stem cells(PDLSCs).Methods PDLSCs were isolated and cultured using the enzyme digestion method.Treg cells were isolated from human peripheral blood using flow cytometry.Exosomes derived from Treg cells were extracted using the ultracentrifugation method,and identified by transmission electron microscope(TEM),nanosight tracking analysis(NTA)and western blot assay.Different concentrations of Treg cell-derived exosomes(0,1×10^(8),1×10^(10)particles/ml)were added to PDLSCs for co-culture.Real-time RT-PCR and Western Blot were used to detect the expression of Runx2 and Osterix mRNA and protein in PDLSCs after 7 and 14 days of osteogenic induction.The osteogenic mineralization of PDLSCs was analyzed using alizarin red staining.Western blot was used to examine the expression of Hes-1 protein in PDLSCs before and after treatment with Treg cell-derived exosomes.Results Treg cellderived exosomes exhibited a circular or elliptical shape with an average diameter of approximately 139nm.Western blot analysis confirmed the presence of CD9,CD63,CD81,and TSG101 proteins in the extracted exosomes.Treg cell-derived exosomes promoted the expression of Runx2 and Osterix mRNA and protein in PDLSCs.Compared to the control group,PDLSCs were able to form many calcium nodules in the Treg cell-derived exosome group.Moreover,the expression of Hes-1 protein in PDLSCs was upregulated in Treg cell-derived exosome group.Conclusion Treg cell-derived exosomes can enhance the osteogenic differentiation of PDLSCs,potentially through the activation of the Notch signaling pathway.