三氯生对牙髓干细胞生物学特性的影响  

Effects of triclosan on the biological characteristics of dental pulp stem cells

作　　者：王辛鑫 贺继辉 李纲[1,3] 叶青松 贺燕 WANG Xinxin;HE Jihui;LI Gang;YE Qingsong;HE Yan(Institute of Regenerative and Translational Medicine,Affiliated Tianyou Hospi-tal,Wuhan University of Science and Technology,Wuhan 430064,China;First Clinical College of the Ministry of Medicine,Wuhan University of Science and Technology,Wuhan 430064,China;Department of Stomatology,Affiliated Tianyou Hospital,Wuhan University of Science and Technology,Wuhan 430064,China;Center of Regenerative Medicine,Renmin Hospital of Wuhan University,Wuhan University,Wuhan 430060,China;Department of Stomatology,Renmin Hospital of Wuhan University,Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉科技大学附属天佑医院再生与转化医学研究所,湖北武汉430064 [2]武汉科技大学医学部第一临床学院,湖北武汉430064 [3]武汉科技大学附属天佑医院口腔科,湖北武汉430064 [4]武汉大学人民医院再生医学中心,湖北武汉430060 [5]武汉大学人民医院口腔科,湖北武汉430060

出　　处：《口腔疾病防治》2024年第11期834-844,共11页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：湖北省重点研发计划项目(2022BCA029);湖北省卫生健康委科研项目(WJ2023M121);口腔颌面发育与再生湖北省重点实验室开放基金(2022kqhm005);武汉科技大学楚天学者计划(X22020024)。

摘　　要：目的探讨环境污染物三氯生(triclosan,TCS)对牙髓干细胞(dental pulp stem cells,DPSCs)的多种生物学特性是否有消极影响,以及TCS在大鼠体内牙髓组织的分布情况和危害,为DPSCs的临床应用和TCS的安全性提供依据。方法获得武汉科技大学附属天佑医院医学伦理委员会批准,收集离体牙,分离、培养并鉴定人来源的DPSCs,在DPSCs体外培养液中加入0~0.08 mmol/L的TCS,通过CCK-8检测DPSCs的增殖能力,划痕实验检测DPSCs的迁移能力,诱导三系分化检测DPSCs的分化能力,检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、转化生长因子-β(transforming growth fac-tor-β,TGF-β)的基因或蛋白表达,荧光染色分析DPSCs生成活性氧(reactive oxygen species,ROS)的水平,荧光探针检测DPSCs线粒体膜电位的改变,以及检测DPSCs的PI3K/Akt/mTOR、p38和JNK通路活性。获得武汉科技大学实验动物伦理委员会批准,建立50 mg/kg/d的TCS短期灌胃暴露2个月的大鼠模型,收集并通过液相色谱-质谱联用法检测大鼠肝脏、大脑和牙髓组织中的TCS浓度。结果0.02 mmol/L、0.04 mmol/L和0.08 mmol/L的TCS在与人来源DPSCs接触的第5天和第7天显著抑制了其增殖能力;0.04 mmol/L和0.08 mmol/L的TCS在接触第3天显著抑制了DPSCs的迁移能力和三系分化能力,诱导了促炎因子TNF-α、IL-1β、IL-6、iNOS以及TGF-β的基因或蛋白表达,抑制了抗炎因子IL-10的蛋白表达;0.04 mmol/L和0.08 mmol/L的TCS在接触第1天诱导了DPSCs的ROS产生,降低了DPSCs的线粒体膜电位,并在第3天抑制了DPSCs的PI3K/Akt/mTOR通路活性,增强了p38通路活性,不影响JNK通路活性;大鼠短期TCS灌胃暴露后,在肝脏(430ng/mL)和大脑(41.4ng/mL)组织中检测到TCS存在,牙髓中未检测到,TCS分布浓度最高的Objective To explore whether the environmental pollutant triclosan(TCS)has negative effects on the various biological characteristics of dental pulp stem cells(DPSCs),as well as the distribution and hazards of TCS in rat dental pulp tissue in vivo,which will provide a basis for the clinical application of DPSCs and the safety of TCS.Methods Tooth collection was approved by the Ethics Committee of Tianyou Hospital Affiliated to Wuhan University of Science and Technology.Human DPSCs were extracted,cultured,and identified.Up to 0.08 mmol/L of TCS was added to the in vitro culture medium of DPSCs.The proliferation ability of DPSCs was detected by CCK-8.The migration ability of DPSCs was detected via scratch assay.The differentiation ability of DPSCs was detected by inducing trilineage differenti-ation.The gene or protein expression levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-10(IL-10),inducible nitric oxide synthase(iNOS),and transforming growth factor-β(TGF-β)in DPSCs were detected.The level of reactive oxygen species(ROS)generated by DPSCs was analyzed using fluorescence staining.Changes in mitochondrial membrane potential of DPSCs were detected using a fluorescent probe.The activity of PI3K/Akt/mTOR,p38,and JNK pathways of DPSCs were detected.Animal experiments were approved by the Animal Ethics Committee of Wuhan University of Science and Technology.A rat model of short-term oral exposure to 50 mg/kg/d of TCS for 2 months was established,and the TCS concentration in the liver,brain,and dental pulp tissues of rats was detected through liquid chromatography-mass spectrometry.Results TCS at 0.02 mmol/L,0.04 mmol/L,and 0.08 mmol/L significantly inhibited the proliferation ability of human-derived DPSCs on the 5th and 7th days of contact.TCS at 0.04 mmol/L and 0.08 mmol/L significantly inhibited the migration ability and tri-lineage differentiation ability of DPSCs on the 3rd day of contact.TCS induced the gene or protein expression of proinflammatory factors in

关 键 词：三氯生 牙髓干细胞 增殖 分化 活性氧 氧化应激 炎性因子 线粒体膜电位 PI3K/AKT/MTOR 牙髓组织 

分 类 号：R78[医药卫生—口腔医学]