M2型巨噬细胞通过调控氧化-抗氧化体系和线粒体自噬影响人牙周膜干细胞的成牙骨质分化潜能  

M2 Macrophages regulate cementogenic differentiation of human periodontal ligament stem cells by modulating oxidant-antioxidant system and mitophagy

作　　者：甘典 陈发明 李璇 GAN Dian;CHEN Faming;LI Xuan(State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi International Joint Research Center for Oral Diseases,Department of Periodontology,The Third Hospital Affiliated of Air Force Military Medical University,China,Xi'an 710032)

机构地区：[1]口颌系统重建与再生全国重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔疾病国际联合研究中心,空军军医大学第三附属医院牙周病科,西安710032

出　　处：《实用口腔医学杂志》2024年第2期164-172,共9页Journal of Practical Stomatology

基　　金：国家自然科学基金(编号:82301079);中国科协青年人才托举工程(编号:2022QNRC001)。

摘　　要：目的:探索巨噬细胞(Mφ)极化对人牙周膜干细胞(hPDLSCs)成牙骨质分化的影响及作用机制。方法:将THP-1人单核细胞分别诱导为M0、M1和M2型Mφ,用RPMI 1640基础培养基或不同极化状态Mφ来源的上清液与成牙骨质诱导液等比例混合制备条件培养基(CM-Control、CM-M0、CM-M1和CM-M2)用于培养hPDLSCs,将条件培养基培养的hPDLSCs形成的细胞膜片包裹人脱矿牙本质基质(TDM)移植至裸鼠皮下进行异位牙骨质形成实验。以CM-Control组作为对照,通过HE染色实验观察类牙骨质样组织的形成;免疫荧光染色(IMF)和qRT-PCR,检测成牙骨质分化标记物骨涎蛋白(BSP)、牙骨质附着蛋白(CAP)和牙骨质蛋白-1(CEMP-1),氧化-抗氧化体系标记物超氧化物歧化酶1(SOD1)和核转录因子红系2相关因子2(NRF2)以及线粒体自噬标记物PTEN诱导假定激酶1(PINK1)和微管相关蛋白1A/1B-轻链3(LC3)的表达水平。结果:体内实验中,CM-M2组形成的类牙骨质样组织更多,CAP、CEMP-1蛋白表达量更高且伴随SOD1蛋白和PINK1、LC3蛋白表达水平升高,NRF2蛋白未见明显差异。体外实验中,CM-M2组BSP(P<0.01)、CAP(P<0.001)和CEMP-1(P<0.01)的mRNA水平上升,SOD1的mRNA水平上升(P<0.05),而NRF2的mRNA水平无统计学差异(P>0.05),PINK1的mRNA水平上升(P<0.05)。结论:M2型Mφ可能通过上调hPDLSCs的氧化-抗氧化体系和线粒体自噬水平促进hPDLSCs的成牙骨质分化潜能。Objective:To investigate the effects of macrophage(Mφ)polarization on the cementogenic differentiation of human periodontal ligament stem cells(hPDLSCs)and the underlying mechanism.Methods:Human monocytic THP-1 cells were induced to M0,M1 and M2 Mφsubsets,then RPMI 1640 medium or supernatants of different Mφphenotypes were mixed with an equal volume of cementoblastic induction medium to generate conditioned mediums(CMs),and termed as CM-Control,CM-M0,CM-M1 and CM-M2,respectively.hPDLSCs were cultured with different CMs,and the hPDLSCs sheets were then wrapped around treated dentin matrix(TDM)to generate cell sheet/dentin complexes.The complexes were subcutaneously implanted into nude mice.The cementum-like tissue formation was evaluated by HE staining,immunofluorescent staining(IMF)and qRT-PCR were used to detect the expression level of cementogenic differentiation-related markers bone sialoprotein(BSP),cementum attachment protein(CAP)and cementum protein-1(CEMP-1),oxidant-antioxidant system-related markers superoxide dismutase 1(SOD1)and nuclear factor erythroid 2-related factor 2(NRF2),mitophagy-related markers PTEN induced putative kinase 1(PINK1)and microtubule asso ciated proteins 1A/1B light chain 3(LC3).Results:In vivo,CM-M2-treated hPDLSCs(CM-M2)group formed more cementum-like tissues and expressed higher protein levels of CAP,CEMP-1,SOD1,PINK1 and LC3 than that in other groups.In vitro tests showed that,compared with CM-Control group,hPDLSCs incubated with CM-M2 increased the levels of BSP(P<0.01),CAP(P<0.001),CEMP-1(P<0.01)and SOD1(P<0.05),while no statistically significant difference was detected for NRF2(P>0.05),and increasedthe expression of PINK1(P<0.05).Conclusion:M2 Mφregulate the cementogenic differentiation of hPDLSCs possibly via modulating oxidant-antioxidant system and mitophagy.

关 键 词：成牙骨质分化 巨噬细胞极化 牙周膜干细胞 线粒体自噬 

分 类 号：R781.4[医药卫生—口腔医学]