摘要
背景:近年来牙周膜干细胞外泌体在牙周组织再生工程中的应用得到了广泛研究,但其对于炎症环境来源牙周膜干细胞的影响尚不清楚。目的:探讨健康和炎症环境来源牙周膜干细胞所分泌的外泌体对炎症环境来源牙周膜干细胞增殖及分化能力的影响。方法:采用酶消化法分离培养健康牙周组织与牙周炎组织来源的人牙周膜干细胞,采用超速离心法提取两种来源人牙周膜干细胞的外泌体。选取生长状态良好的第3代炎症组织来源牙周膜干细胞,分3组培养:空白组常规培养,健康外泌体组加入健康组织来源外周膜干细胞分泌的外泌体,炎症外泌体组加入炎症组织来源人牙周膜干细胞分泌的外泌体,检测细胞增殖与克隆形成;在成骨诱导分化条件下,检测细胞碱性磷酸酶表达与矿化结节形成、成骨相关基因mRNA与蛋白的表达。结果与结论:①CCK-8检测与克隆形成实验显示,与空白组相比,两种来源外泌体均可促进炎症组织来源牙周膜干细胞的增殖与细胞集落形成(P<0.05),并且健康外泌体组的作用强于炎症外泌体组(P<0.05);②碱性磷酸酶与茜素红染色显示,与空白组相比,两种来源外泌体均可促进炎症组织来源牙周膜干细胞碱性磷酸酶的表达与矿化结节形成,并且健康外泌体组的促进作用强于炎症外泌体组;RT-PCR与Western Blot检测显示,与空白组相比,两种来源外泌体均可促进炎症组织来源牙周膜干细胞碱性磷酸酶、RUNX2、Ⅰ型胶原mRNA与蛋白的表达(P<0.05),并且健康外泌体组的促进作用强于炎症外泌体组(P<0.05);③结果表明,人牙周膜干细胞分泌的外泌体能够促进炎症环境来源牙周膜干细胞的增殖及成骨分化能力,并且健康组织来源人牙周膜干细胞分泌外泌体的促进作用优于炎症组织来源人牙周膜干细胞分泌的外泌体。
BACKGROUND:In recent years,the application of exosomes of periodontal ligament stem cells in periodontal tissue regeneration engineering has been widely studied,but the effect of exosomes on periodontal ligament stem cells derived from inflammatory environment is still unclear.OBJECTIVE:To investigate the effects of exosomes secreted by periodontal ligament stem cells from healthy and inflammatory environments on the proliferation and differentiation of periodontal ligament stem cells from inflammatory environments.METHODS:Human periodontal ligament stem cells from healthy and inflammatory tissues were isolated and cultured by enzyme digestion method.Exosomes were extracted from two kinds of periodontal ligament stem cells using ultracentrifugation.Passage 3 periodontal ligament stem cells derived from inflammatory tissue were selected and cultured in three groups.Cells in the blank group were cultured routinely.The healthy exosome group was added with exosomes secreted by peripheral ligament stem cells derived from healthy tissue.The inflammatory exosome group was added with exosomes secreted by human periodontal ligament stem cells derived from inflammatory tissue.Cell proliferation and cloning were detected.The expression of alkaline phosphatase,the formation of mineralized nodules,and the expression of mRNA and protein of genes related to osteogenesis were detected under osteogenic differentiation.RESULTS AND CONCLUSION:(1)CCK-8 assay and clonal formation test showed that compared with the blank group,two kinds of exosomes could promote the proliferation and colony formation of periodontal ligament stem cells from inflammatory tissue(P<0.05),and the effect of the healthy exosome group was stronger than that of the inflammatory exosome group(P<0.05).(2)Alkaline phosphatase and alizarin red staining showed that compared with the blank group,the two kinds of exosomes could promote the expression of alkaline phosphatase and the formation of mineralized nodules in periodontal ligament stem cells from inflammatory tissue,and the promoting effect of the healthy exosome group was stronger than that of the inflammatory exosome group.RTPCR and western blot assay showed that compared with the blank group,the two kinds of exosomes could promote the expression of alkaline phosphatase,RUNX2,and type I collagen mRNA and protein in periodontal ligament stem cells from inflammatory tissue(P<0.05).The promoting effect of the healthy exosome group was stronger than that of the inflammatory exosome group(P<0.05).(3)The results showed that exosomes secreted by human periodontal ligament stem cells could promote the proliferation and osteogenic differentiation of periodontal ligament stem cells derived from inflammatory environments,and the promoting effect of exosomes secreted by human periodontal ligament stem cells derived from healthy tissues was better than that from human periodontal ligament stem cells derived from inflammatory tissues.
作者
蒋志梁
罗雅馨
胡峥祺
杨丽
杨婵婵
陈虹
刘小艺
黄艳
杨琨
Jiang Zhiliang;Luo Yaxin;Hu Zhengqi;Yang Li;Yang Chanchan;Chen Hong;Liu Xiaoyi;Huang Yan;Yang Kun(Stomatological Hospital of Zunyi Medical University,Zunyi 563006,Guizhou Province,China;Department of Periodontology,Affiliated Stomatological Hospital of Zunyi Medical University,Zunyi 563006,Guizhou Province,China;Zunyi Medical University,Zunyi 563006,Guizhou Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第13期2744-2752,共9页
Chinese Journal of Tissue Engineering Research
基金
贵州省卫健委科学技术基金项目(gzwkj2022-423),项目名称:ROS/NLRP3信号通路在2型糖尿病伴牙周炎患者PMN自噬介导炎症反应中的调控机制
贵州省卫健委科学技术基金项目(gzwkj2022-169),项目名称:P75NTR阳性的牙槽窝来源的间充质干细胞的生物学特性的研究,项目负责人:杨琨
贵州省科学技术基金计划(黔科合基础[2020]1Y328),项目名称:羊膜匀浆对牙周膜干细胞增殖分化的影响及PLA/PCL纤维膜增强羊膜负载对大鼠牙周缺损修复的研究,项目负责人:杨琨
贵州省科技厅基础研究项目(黔科合基础-ZK[2023]一般535),项目名称:负载EX-4和SDF-1的PLGA静电纺丝纤维膜促进PDLSCs增殖、迁移、成血管及成神经分化的作用和机制研究
合肥医科大学附属医院院基金[院字(2022)02号],项目名称:2型糖尿病伴牙周炎牙周膜干细胞外泌体对颌骨骨髓间充质细胞成骨分化的影响研究,项目负责人:杨琨。
关键词
牙周炎
干细胞
牙周膜干细胞
外泌体
细胞增殖
成骨分化
组织工程
periodontitis
stem cell
periodontal ligament stem cell
exosome
cell proliferation
osteogenic differentiation
tissue engineering
