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

作　　者：刘浩天 闫福华[1] 伍玉 童昕[1] 张倩[1] LIU Haotian;YAN Fuhua;WU Yu;TONG Xin;ZHANG Qian(Nanjing Stomatological Hospital,Affiliated Hospital of Medical School,Research Institute of Stomatology,Nanjing University,Nanjing 210008,China)

机构地区：[1]南京大学医学院附属口腔医院,南京市口腔医院,南京大学口腔医学研究所,江苏南京210008

出　　处：《口腔疾病防治》2025年第4期268-277,共10页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金(82201065);南京市卫生科技发展专项资金项目(YKK21180);南京大学医学院附属口腔医院“3456”育才计划(0222R214);江苏省医学重点学科/实验室建设单位(JSDW202246)。

摘　　要：目的 研究牙周膜干细胞(periodontal ligament stem cells,PDLSCs)来源的细胞内囊泡(intracellular vesicles,IVs)、细胞外囊泡(extracellular vesicles,EVs)对脂多糖(lipopolysaccharide,LPS)模拟的炎症微环境下PDLSCs成骨分化的影响,为IVs在牙周炎组织修复与再生的应用提供新的思路。方法 获得单位伦理审批,提取人来源的PDLSCs,收集第3~6代PDLSCs来源的IVs、EVs,使用透射电镜、纳米流式分析、Western Blot对其鉴定。取第3~6代PDLSCs分为:Control组、LPS组、LPS+100μg/mL EVs组(LPS+EVs组)和LPS+100μg/mL IVs组(LPS+IVs组)。通过CCK-8、酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)、实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)、Western Blot、碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色(alizarin red staining,ARS)评估炎症微环境下IVs、EVs对PDLSCs抗炎和促成骨分化的影响。结果 透射电镜下PDLSCs来源的EVs、IVs呈现双层膜结构,纳米流式分析显示IVs、EVs的平均粒径分别为79.6 nm、82.1 nm,Western Blot检测IVs、EVs表面蛋白CD9、CD63、CD81表达阳性,Calnexin表达阴性,IVs、EVs获取成功。相较于Control组,LPS组PDLSCs增殖降低,炎症因子白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平增高,成骨分化相关基因Runt相关转录因子2(runt-related transcription factor 2,RUNX2)、碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)mRNA表达降低,成骨分化相关蛋白RUNX2和骨桥蛋白(osteopontin,OPN)蛋白表达降低,差异有统计学意义(P<0.05);相较于LPS组,LPS+EVs组和LPS+IVs组PDLSCs增殖增高,炎症因子IL-6、TNF-α水平降低,RUNX2、ALP、OCN的mRNA表达增高,成骨分化相关蛋白RUNX2和OPN蛋白表达增高,差异有统计学意义(P<0.05);且炎症微环境下,IVs促进PDLSCs增殖、抑制TNF-α、促进RUNX2 mRNA表达、促进RUNX2蛋白和OPN蛋白表达,促进ALP活性及矿化结节�Objective To examine the effect of intracellular vesicles(IVs)and extracellular vesicles(EVs)that originated from periodontal ligament stem cells(PDLSCs)on the osteogenic differentiation of PDLSCs within a lipopolysaccharide(LPS)‐simulated inflammatory microenvironment,and to provide new insights for the application of IVs in the repair and regeneration of periodontal tissue in periodontitis.Methods Ethical approval was obtained from the institution.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+100μg/mL EVs group(LPS+EVs group),and LPS+100μg/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 using 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,respectively.Western Blot analysis indicated that the surface proteins CD9,CD63,and CD81 of the IVs and EVs were positively expressed,while calnexin was negatively expressed,indicating that IVs and EVs were successfully obtained.Compared with the Control group,the proliferation of PDLSCs in the LPS group was reduced,while the levels of inflammatory 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 transcri

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 

分 类 号：R78[医药卫生—口腔医学]