国际口腔医学杂志 ›› 2022, Vol. 49 ›› Issue (1): 27-36.doi: 10.7518/gjkq.2022026
Ai Xiaoqing(),Dou Lei,Qiao Xin,Yang Deqin(
)
摘要:
目的 分析并比较二维(2D)与三维(3D)培养条件下人牙髓间充质细胞(DPSCs)外泌体(Exo)微小RNA(miRNA)的表达谱。方法 2D与3D条件下分别培养DPSCs,提取细胞Exo,采用透射电子显微镜、蛋白质免疫印迹及纳米颗粒追踪分析等方法观察并鉴定;通过高通量测序筛选差异表达miRNA,采用Dr.Tom系统及TargetScan网站进行生物信息学分析及组织再生修复相关靶基因预测。结果 3D培养下收集的DPSC-Exo均呈现“茶托样”双层膜结构,CD63和CD9表达阳性,粒径分布符合外泌体特征,与2D培养的DPSC-Exo一致。高通量测序共计检测外泌体来源miRNA 253个,其中3D组表达222个,特有表达99个;与2D组相比,表达显著差异60个[︱log2(3D/2D)︱≥1,Qvalue≤0.001]。差异miRNA主要参与的生物学过程与分子功能分别为细胞过程和结合;京都基因与基因组数据库(KEGG)通路富集分析显示在代谢通路有显著富集。miR-302的候选靶基因有成纤维细胞生长因子(FGF)19和表皮生长因子受体等;miR-24-3p可能靶向神经元分化因子2、神经上皮细胞转化因子1、神经元分化因子1、神经元再生相关蛋白、FGF11、FGF结合蛋白3、FGF受体3、血小板衍生生长因子受体(PDGFR)β多肽、PDGFRα多肽、血管生成素、胰岛素样生长因子结合蛋白5等,以发挥组织再生修复功能。结论 相比2D培养,3D培养能调节DPSC-Exo部分miRNA表达量,上调一些与再生修复相关的miRNA,3D培养可考虑作为调节外泌体应用潜能的一种手段。
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