人牙周膜干细胞来源外泌体干预成骨细胞MC3T3-E1的增殖和分化

Human periodontal ligament stem cells-derived exosomes interfere with the proliferation and differentiation of MC3T3-E1 cells

背景:人牙周膜干细胞具有较强的成骨分化能力,人牙周膜干细胞来源外泌体作为牙周膜干细胞分泌的主要成分,对成骨细胞MC3T3-E1增殖和成骨分化的影响尚不明确。目的:探讨人牙周膜干细胞来源外泌体对MC3T3-E1细胞增殖和分化的影响。方法:采用酶消化法分离及培养人牙周膜干细胞,超速离心法提取人牙周膜干细胞来源外泌体,通过透射电镜、粒径分析及Western blot方法对人牙周膜干细胞来源外泌体进行鉴定;CCK8法检测不同质量浓度人牙周膜干细胞来源外泌体对MC3T3-E1细胞增殖的影响,茜素红染色观察100 mg/L人牙周膜干细胞来源外泌体对MC3T3-E1细胞成骨矿化的影响,Western blot检测100 mg/L人牙周膜干细胞来源外泌体干预前后MC3T3-E1细胞内MEK和ERK的磷酸化水平。结果与结论:①透射电镜观察可见外泌体为脂质双分子层形成的囊泡结构,粒径检测显示外泌体直径分布在50-120 nm,集中在79.86 nm,Western blot检测结果显示提取的外泌体中含有CD81,CD63,TSG101的表达;②与对照组相比,人牙周膜干细胞来源外泌体对MC3T3-E1细胞的增殖具有促进作用,且作用呈剂量依赖性;③与对照组相比,人牙周膜干细胞来源外泌体组MC3T3-E1细胞能够形成更多的钙结节;与对照组相比,人牙周膜干细胞来源外泌体组MC3T3-E1细胞内p-MEK及p-ERK蛋白表达量升高;④结果表明,人牙周膜干细胞来源外泌体可以显著促进MC3T3-E1增殖和成骨分化,推测可能与其激活MEK/ERK信号通路有关。

BACKGROUND:Human periodontal ligament stem cells had a strong osteogenic ability,but the effect of human periodontal ligament stem cells-derived exosomes on proliferation and osteogenic differentiation of MC3T3-E1 is not clear.OBJECTIVE:To explore the effect of human periodontal ligament stem cells-derived exosomes on the proliferation and differentiation of MC3T3-E1 cells.METHODS:Human periodontal ligament stem cells were isolated and cultured by enzyme digestion.Human periodontal ligament stem cells-derived exosomes were extracted by ultracentrifugation,and identified by transmission electron microscopy,nanoparticle tracking analysis and western blot assay.Effect of different mass concentrations of human periodontal ligament stem cells-derived exosomes on proliferation of MC3T3-E1 cells was detected by CCK-8 assay.The effect of 100 mg/L human periodontal ligament stem cells-derived exosomes on osteogenic mineralization of MC3T3-E1 cells was observed by alizarin red staining.Western blot assay was used to detect the phosphorylation levels of MEK and ERK in MC3T3-E1 cells before and after the intervention of 100 mg/L human periodontal ligament stem cells-derived exosomes.RESULTS AND CONCLUSION:(1)Transmission electron microscopy showed that exosomes were vesicles formed by lipid bilayer.Nanoparticle tracking analysis showed that exosomes were 50-120 nm in diameter and concentrated at 79.86 nm.Western blot assay showed that the exosomes contained expression levels of CD81,CD63 and TSG101.(2)Compared with the control group,human periodontal ligament stem cells-derived exosomes could promote the proliferation of MC3T3-E1 cells in a dose-dependent manner.(3)Compared with the control group,MC3T3-E1 cells were able to form many calcium nodules in the human periodontal ligament stem cell-derived exosome group.Compared with the control group,protein expression of p-MEK and p-ERK increased in MC3T3-E1 cells of the human periodontal ligament stem cell-derived exosome group.(4)It is indicated that human periodontal ligament stem cells-derived exosomes can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells,which may be associated with the activation of MEK/ERK signaling pathway.