牙龈卟啉单胞菌外膜囊泡通过RgpB抑制骨髓间充质干细胞成骨向分化能力  

Porphyromonas gingivalis outer membrane vesicles inhibit osteogenic differentiation of bone marrow mesenchymal stem cells through RgpB

作　　者：罗醒红 王妍珍 麻丹丹[1] LUO Xinghong;WANG Yanzhen;MA Dandan(Department of Endodontics,Stomatological Hospital,Southern Medical University,Stomatological College of Southern Medical University,Guangzhou 510280,China)

机构地区：[1]南方医科大学口腔医学院·南方医科大学口腔医院牙体牙髓科,广东广州510280

出　　处：《口腔生物医学》2025年第1期6-13,共8页Oral Biomedicine

基　　金：国家自然科学基金(82470963);南方医科大学口腔医院科研培育计划(PY2023027)。

摘　　要：目的:探究牙龈卟啉单胞菌(P.g)外膜囊泡(OMVs)通过精氨酸特异性牙龈蛋白酶B(RgpB)分子对骨髓间充质干细胞(BMSCs)成骨功能的影响。方法:分离鉴定P.g OMVs,与BMSCs共培养,通过实时荧光定量PCR、Western blot检测碱性磷酸酶(ALP)、骨形成蛋白2(BMP2)及骨钙素(OCN)基因和蛋白的变化;利用蛋白酶K处理的P.g OMVs去除其蛋白成分后,与BMSCs共培养,Western blot、ALP染色及茜素红染色检测其成骨能力的变化,验证P.g OMVs是否通过其携带的蛋白分子抑制BMSCs成骨能力;进一步利用液相色谱-质谱分析P.g OMVs的蛋白成分,筛选出可能的蛋白分子RgpB,并利用实时荧光定量PCR及ALP染色验证其对BMSCs成骨向分化能力的影响。结果:实时荧光定量PCR和Western blot结果均显示,P.g OMVs抑制BMSCs中ALP、BMP2及OCN基因和蛋白水平的表达;去除P.g OMVs中蛋白分子,BMSCs中ALP、BMP2蛋白水平的表达升高,ALP活性及矿化结节形成能力增强。液相色谱-质谱分析结果表明OMVs中高表达RgpB分子。利用RgpB纯化蛋白刺激BMSCs,随着RgpB浓度的升高,BMSCs中ALP、BMP2及OCN基因的表达降低(P<0.05),ALP活性受到抑制。结论:P.g OMVs可通过携带RgpB抑制BMSCs成骨向分化能力。Objective:To investigate the effect of Porphyromonas gingivalis(P.g) outer membrane vesicles(OMVs) on bone marrow mesenchymal stem cells(BMSCs) osteogenesis through arginine-specific gingipain B(RgpB).Methods:P.g OMVs were isolated and identified,co-cultured with BMSCs.The changes in the gene and protein levels of alkaline phosphatase(ALP),bone morphogenetic protein 2(BMP2),and osteocalcin(OCN) were detected by real-time fluorescent quantitative PCR(qRT-PCR) and Western blot.To investigate the effect of proteins in P.g OMVs on BMSCs osteogenic differentiation,P.g OMVs were treated with proteinase K to remove their protein components and then co-cultured with BMSCs,and Western blot,ALP staining,and alizarin red staining were used to examine changes in osteogenic ability.The results verified whether P.g OMVs inhibit BMSCs osteogenesis through the proteins they carry.Further,liquid chromatography-mass spectrometry analysis of P.g OMVs was conducted to screen for potential protein molecules,RgpB.The effect of RgpB on the osteogenic differentiation ability of BMSCs was verified by qRT-PCR and ALP staining.Results:Both qRT-PCR and Western blot results showed that P.g OMVs inhibited the expression of ALP,BMP2,and OCN genes and proteins in BMSCs.When the protein molecules in P.g OMVs were removed,the expression of ALP and BMP2 proteins in BMSCs increased,along with enhanced ALP activity and mineralized nodule formation.Mass spectrometry analysis revealed high expression of RgpB in the OMVs.When BMSCs were stimulated with RgpB protein,the expression of ALP,BMP2,and OCN genes in BMSCs decreased as RgpB concentration increased(P<0.05),and ALP activity was suppressed.Conclusion:P.g OMVs can inhibit the osteogenic differentiation of BMSCs by carrying the RgpB.

关 键 词：骨髓间充质干细胞 牙龈卟啉单胞菌 外膜囊泡 成骨向分化 RgpB 

分 类 号：R780.2[医药卫生—口腔医学]