间充质干细胞来源凋亡囊泡缓解牙龈卟啉单胞菌脂多糖诱导下巨噬细胞促炎状态的研究  

Mesenchymal stem cells derived apoptotic extracellular vesicles attenuate pro-inflammatory macrophages induced by Porphyromonas gingivalis lipopolysaccharide

作　　者：叶庆元 李子涵 王垭铮 刘世宇[2] 周峻[2] 刘思颖[3] 王勤涛[1] Ye Qingyuan;Li Zihan;Wang Yazheng;Liu Shiyu;Zhou Jun;Liu Siying;Wang Qintao(Department of Periodontology,School of Stomatology,The Fourth Military Medical University&State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture,Xi′an 710032,China;Department of Oral Histopathology,School of Stomatology,The Fourth Military Medical University&State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi International Joint Research Center for Oral Diseases,Xi′an 710032,China;Department of Orthodontics,School of Stomatology,The Fourth Military Medical University&State Key Laboratory of Military Stomatology&National Clinical Research Center for Oral Diseases&Shaanxi Clinical Research Center Research Center for Oral Diseases,Xi′an 710032,China)

机构地区：[1]第四军医大学口腔医学院牙周病科,军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔生物工程技术研究中心,西安710032 [2]第四军医大学口腔医学院口腔组织病理学教研室,军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔疾病国际联合研究中心,西安710032 [3]第四军医大学口腔医学院正畸科,军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔疾病临床医学研究中心,西安710032

出　　处：《中华口腔医学杂志》2021年第8期791-798,共8页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81771069,31870970,31800817)。

摘　　要：目的探索骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSC)来源凋亡囊泡对小鼠巨噬细胞系RAW264.7极化状态的影响及对牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)诱导的促炎状态巨噬细胞的调控,为牙周炎治疗提供依据。方法使用Operetta CLS高内涵分析系统动态观察星形孢菌素(staurosporine,STS)诱导的BMMSC的凋亡过程;使用场发射扫描电镜、动态光散射技术、流动电势法测量凋亡囊泡相关表征;使用Operetta CLS高内涵分析系统动态观察小鼠巨噬细胞系RAW264.7对5-羧基四甲基罗丹明琥珀酰亚胺酯(5-carboxy-tetramethylrhodamine,succinimidyl ester,5-TAMRA-SE)标记的凋亡囊泡的吞噬作用;采用实时荧光定量PCR检测精氨酸合酶-1(arginase-1,Arg-1)mRNA相对表达量;采用细胞免疫荧光、蛋白质印迹法检测凋亡囊泡对Pg来源的脂多糖(lipopolysaccharide from Pg,Pg-LPS)诱导的RAW264.7中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达的影响;采用酶联免疫吸附测定法检测Pg-LPS诱导的促炎状态RAW264.7培养上清液中肿瘤坏死因子-α(tumor necrosis factro-α,TNF-α)的分泌情况。结果0.5μmol/L STS处理12 h后小鼠BMMSC形态皱缩,周围有明显的凋亡囊泡产生;凋亡囊泡的粒径为100~1000 nm,平均Zeta电势为-16.6 mV;RAW264.7可以通过伪足吞噬小鼠BMMSC来源的凋亡囊泡;小鼠BMMSC来源的凋亡囊泡联合白介素-4(interleukin4,IL-4)刺激的RAW 264.7 Arg-1 mRNA表达量(261.97±15.91)较单独IL-4刺激的mRNA表达量(115.29±15.42)显著升高(P<0.01);凋亡囊泡与Pg-LPS共同作用下,RAW 264.7 iNOS阳性细胞数量[(39.33±4.70)个]显著低于Pg-LPS单独诱导的RAW 264.7 iNOS阳性细胞数量[(95.33±4.70)个](P=0.007),RAW264.7 iNOS蛋白相对表达量(5.84±1.05)显著低于Pg-LPS单独诱导的RAW 264.7 iNOS蛋白相对表达量(14.91±3.87)(P<0.01),RAW264.7细胞培养上清液中TNF-α含量[(21899.71±409.73)ng/L]显著低于Pg-LPS单独诱导下RAW264.7细胞�Objective To investigate whether bone marrow mesenchymal stem cells(BMMSCs)derived apoptotic extracellular vesicles(ApoEVs)could regulate the polarization of mouse macrophage cell line RAW264.7 and whether BMMSCs derived ApoEVs could attenuate pro-inflammatory condition of RAW264.7 induced by Porphyromonas gingivalis lipopolysaccharide(Pg-LPS),so as to provide experimental evidence and theoretical basis for using BMMSCs derived ApoEVs as a method to treat periodontitis.Methods The Operetta CLS high-content analysis system was used to observe the time-dependent apoptosis process of BMMSCs.Besides,field emission scanning electron microscopy(FESEM),dynamic light scattering technology and streaming potential method were used to measure the surface characteristics of BMMSCs derived ApoEVs.The Operetta CLS high-content analysis system was used to observe the process of RAW264.7 phagocyting 5-carboxy-tetramethylrhodamine,succinimidyl ester(5-TAMRA-SE)labeled ApoEVs.Real-time quantitative PCR was used to detect the mRNA expression of arginase-1(Arg-1).Cell immunofluorescence and Western blotting were used to detect the number of inducible nitric oxide synthase(iNOS)(+)macrophages and iNOS protein expression level in each experiment group.Enzyme linked immunosorbent assay was used to detect tumor necrosis factro-α(TNF-α)level in the Pg-LPS induced pro-inflammatory macrophage culture supernatant in each experiment group.Results After treating with 0.5μmol/L staurosporine for 12 hours,mouse BMMSCs underwent shrinking with obvious vesicles structure around.The FESEM showed the ApoEVs were in spherical shapes.The size range of ApoEVs was about 100-1000 nm and the average Zeta potential was-16.6 mV.The Operetta CLS high-content analysis system showed RAW264.7 could phagocytose 5-TAMRA-SE labeled ApoEVs by pseudopodia.The relative mRNA expression of Arg-1 was significantly increased in RAW 264.7 after being treated with interleukin 4(IL-4)and ApoEVs(261.97±15.91)compared to that with IL-4 alone(115.29±15.42)(P<0.01).Cell i

关 键 词：牙周炎 间质干细胞 巨噬细胞 凋亡囊泡 极化状态 

分 类 号：R781.42[医药卫生—口腔医学]