血管生成素4对牙髓干细胞成牙本质向分化的作用  

Effect of angiopoietin 4 on odontogenic differentiation of dental pulp stem cells

作　　者：范心怡 刘苍维 周怡君 任飞龙 史册[1,2] 孙宏晨 FAN Xinyi;LIU Cangwei;ZHOU Yijun;REN Feilong;SHI Ce;SUN Hongchen(Department of Pathology,Stomatological Hospital of Jilin University,Changchun 130021,China;Key Laboratory of Tooth Development and Jaw Remodeling and Regeneration of Jilin Province,Changchun 130021,China;Department of Pathology,Stomatological Hospital,Medical University,Shenyang 110001,China)

机构地区：[1]吉林大学口腔医院病理科,吉林长春130021 [2]吉林省牙发育及颌骨重塑与再生重点实验室,吉林长春130021 [3]中国医科大学附属口腔医院口腔病理科,辽宁沈阳110001

出　　处：《口腔疾病防治》2023年第10期692-700,共9页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金项目(81970903、81920108012、82270959);吉林省自然科学基金项目(2020020201527JC);吉林省卫生健康青年科技骨干培养计划项目(2019Q013)。

摘　　要：目的探讨血管生成素4(angiopoietin 4,ANGPT4)对牙髓干细胞成牙本质向分化的影响。方法本实验研究已通过单位伦理委员会审查批准,并获得患者知情同意。将人前磨牙进行固定、脱钙、脱水、包埋和切片,免疫荧光染色观察ANGPT4的表达及定位。体外分离培养人牙髓干细胞(human dental pulp stem cells,hDPSCs),于倒置相差显微镜下观察hDPSCs的生长状态及形态;流式细胞术检测细胞表面相关分子标志物的表达;碱性磷酸酶和茜素红S染色鉴定hDPSCs成牙本质向分化的潜能;油红O染色鉴定hDPSCs成脂向分化潜能。提取hDPSCs成牙本质向诱导后不同时间点的RNA,采用RT-qPCR分析hDPSCs体外成牙本质向分化过程中ANGPT4基因及成牙本质细胞相关基因的表达。采用siRNA基因沉默技术沉默hDPSCs中ANGPT4基因的表达,通过RT-qPCR和Western blot检测沉默效率;在hDPSCs中沉默ANGPT4基因表达24 h后进行成牙本质向诱导,在诱导的第7天和14天分别进行碱性磷酸酶和茜素红S染色,检测沉默ANGPT4后hDPSCs的成牙本质向分化能力。结果人前磨牙的免疫荧光染色结果显示,ANGPT4在成牙本质细胞及成牙本质细胞下层多细胞区中表达。hDPSCs经分离培养14 d后状态良好,镜下观察到多个细胞集落,细胞形态均一,呈长梭形;流式细胞术结果显示,hDPSCs表达间充质干细胞标志物CD105(90.42%)和CD90(97.15%),不表达造血细胞标志物CD45(0.01%)和CD34(0.08%)。将hDPSCs进行成牙本质向和成脂向诱导培养后,碱性磷酸酶染色、茜素红S染色和油红O染色均呈阳性。RT-qPCR结果显示,ANGPT4在hDPSCs分化的第5、7、11和14天均高表达(P<0.05),其中第5天表达水平最高。转染si-ANGPT4后,hDPSCs的ANGPT4 mRNA和蛋白表达显著下调(P<0.05);碱性磷酸酶染色结果显示,si-ANGPT4组ALP染色强度和面积均显著低于阴性对照组;茜素红S染色结果显示,si-ANGPT4组钙结节形成明显低于阴性对照组。结论ANGPT4�Objective To investigate the effects of angiopoietin 4(ANGPT4)on the odontogenic differentiation of human dental pulp stem cells.Methods This study has been reviewed and approved by the Ethics Committee,and informed consent has been obtained from patients.Human premolars were fixed,decalcified,dehydrated,embedded,and sectioned.Immunofluorescence staining was used to observe the expression and localization of ANGPT4.Human dental pulp stem cells(hDPSCs)were isolated and cultured in vitro.The growth state and morphology of hDPSCs were observed under an inverted phase contrast microscope.The expression of cell surface-related molecular markers was detected by flow cytometry.Alkaline phosphatase and alizarin red S staining were used to detect the odontogenic differentiation potential of hDPSCs.Oil-red O staining was used to detect the adipogenic differentiation potential of hDPSCs.RNA was extracted from hDPSCs at different time points after odontogenic induction,and RT-qPCR was used to analyze the mRNA expression of ANGPT4 and odontogenic-related genes during the odontogenic differentiation of hDPSCs in vitro.siRNA gene silencing technology was used to silence the expression of ANGPT4 in hDPSCs,and the silencing efficiency was detected by RT-qPCR and Western Blot.After silencing ANGPT4 in hDPSCs for 24 h,odontogenic induction was performed.Alkaline phosphatase and alizarin red S staining were performed on the 7th and 14th of induction to detect the odontogenic differentiation ability of hDPSCs after silencing ANGPT4.Results Immunofluorescence staining of human premolars showed that ANGPT4 was expressed in odontoblasts and sub-odontoblastic cell-rich zone.hDPSCs were in good condition after 14 days of isolation and culture.Under the microscope,multiple cell colonies were observed,and the cell morphology was uniform and long spindle-shaped.The results of flow cytometry showed that hDPSCs expressed mesenchymal stem cell markers CD105(90.42%)and CD90(97.15%),but did not express hematopoietic cell markers CD45(0.01%)and CD

关 键 词：血管生成素4 牙髓干细胞 成牙本质细胞 基因沉默 成牙本质向分化 生长因子 组织再生 牙本质 

分 类 号：R78[医药卫生—口腔医学]