口腔疾病防治2025，Vol.33Issue(4)：268-277,10.DOI:10.12016/j.issn.2096-1456.202440510
牙周膜干细胞来源的内、外囊泡对炎症微环境下牙周膜干细胞成骨分化能力的影响
Effect of intracellular and extracellular vesicles derived from periodontal ligament stem cells on the osteogenic differentiation ability of periodontal ligament stem cells under an inflammatory microenvironment
摘要
Abstract
Objective To examine the effect of intracellular vesicles(IVs)and extracellular vesicles(EVs)that orig-inated from periodontal ligament stem cells(PDLSCs)on the osteogenic differentiation of PDLSCs within a lipopolysac-charide(LPS)-simulated inflammatory microenvironment,and to provide new insights for the application of IVs in the re-pair and regeneration of periodontal tissue in periodontitis.Methods Ethical approval was obtained from the institu-tion.Human-origin PDLSCs were extracted,and the IVs and EVs from PDLSCs at the 3rd-6th passages were gathered and identified using transmission electron microscopy,nano flow cytometry(Nano FCM)analysis,and Western Blot.The 3rd-6th generations of PDLSCs were categorized into the following groups:Control group,LPS group,LPS+100 μg/mL EVs group(LPS+EVs group),and LPS+100 μg/mL IVs group(LPS+IVs group).The effects of the IVs and EVs on the anti-inflammatory and osteogenic differentiation of PDLSCs in an inflammatory microenvironment were assessed by us-ing a Cell Counting Kit-8(CCK-8),enzyme-linked immunosorbent assay(ELISA),quantitative real-time polymerase chain reaction(qRT-PCR),Western Blot,alkaline phosphatase(ALP)staining,and alizarin red staining(ARS).Results Under transmission electron microscopy,the IVs and EVs derived from PDLSCs displayed a double-layer membrane structure.NanoFCM analysis revealed that the average diameters of the IVs and EVs were 79.6 nm and 82.1 nm,respec-tively.Western Blot analysis indicated that the surface proteins CD9,CD63,and CD81 of the IVs and EVs were posi-tively expressed,while calnexin was negatively expressed,indicating that IVs and EVs were successfully obtained.Com-pared with the Control group,the proliferation of PDLSCs in the LPS group was reduced,while the levels of inflammato-ry cytokine interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the cell supernatant were increased,the mRNA expressions of osteogenic differentiation-related genes,including osteoblast-related genes runt-related transcription fac-tor 2(RUNX2),alkaline phosphatase(ALP),osteocalcin(OCN)of PDLSCs were reduced,the protein expressions of RUNX2 and osteopontin(OPN)were also decreased(P＜0.05);compared with the LPS group,the proliferation of PDLSCs in the LPS+EVs group and LPS+IVs group were significantly increased,while the levels of IL-6,TNF-α were significantly reduced,and the mRNA expressions of RUNX2,ALP,OCN were significantly increased,the protein ex-pressions of RUNX2 and OPN were also significantly increased(P＜0.05).Further,in the inflammatory microenviron-ment,Compared with EVs,IVs more significantly promote the proliferation of PDLSCs,inhibit TNF-α expression,en-hance the expression of RUNX2 mRNA,upregulate the expression of RUNX2 and OPN proteins,increase ALP activity,and promote the formation of mineralized nodules(P＜0.05).Conclusion IVs and EVs derived from PDLSCs can boost the proliferation of PDLSCs in an inflammatory microenvironment,inhibit the expression of inflammatory factors,and advance the osteogenic differentiation of PDLSCs.The anti-inflammatory and osteogenic effects of IVs are superior to those of EVs.关键词
牙周膜干细胞/细胞内囊泡/细胞外囊泡/成骨分化/炎症因子/白细胞介素-6/肿瘤坏死因子-α/Runt相关转录因子2/碱性磷酸酶/骨钙素/骨桥蛋白Key words
periodontal ligament stem cells/intracellular vesicles/extracellular vesicles/osteogenic differenti-ation/inflammation factor/interleukin-6/tumor necrosis factor-α/runt-related transcription factor 2/alkaline phosphatase/osteocalcin/osteopontin分类
口腔医学引用本文复制引用
刘浩天,闫福华,伍玉,童昕,张倩..牙周膜干细胞来源的内、外囊泡对炎症微环境下牙周膜干细胞成骨分化能力的影响[J].口腔疾病防治,2025,33(4):268-277,10.基金项目
国家自然科学基金(82201065) （82201065）
南京市卫生科技发展专项资金项目(YKK21180) （YKK21180）
南京大学医学院附属口腔医院"3456"育才计划(0222R214) （0222R214）
江苏省医学重点学科/实验室建设单位(JSDW202246) This study was supported by the grants from National Natural Science Foundation of China(No.82201065) （JSDW202246）
Nanjing Health Science and Technology Development Special Fund Project(No.YKK21180) （No.YKK21180）
"3456"Talent Cultivation Plan of Nanjing Stomatological Hospital,Medical School of Nanjing University(No.0222R214) （No.0222R214）
Jiangsu Provincial Medical Key Discipline(Laboratory)Cultivation Unit(No.JSDW202246). （Laboratory）
