转录因子激活蛋白2C对小鼠磨牙胚发育的影响  

Transcription factor activator protein 2C contribute to molar development in mice

作　　者：刘力维 韩雪 朱辙文 王佐林[1] Liu Liwei;Han Xue;Zhu Zhewen;Wang Zuolin(Department of Oral Implantology,Stomatological Hospital and Dental School of Tongji University,Shanghai Engineering Research Center of Tooth Restoration and Regeneration,Shanghai 200072,China;Department of Pediatric Dentistry,Stomatological Hospital and Dental School of Tongji University,Shanghai Engineering Research Center of Tooth Restoration and Regeneration,Shanghai 200072,China)

机构地区：[1]同济大学口腔医学院·附属口腔医院口腔种植科、上海牙组织修复与再生工程技术研究中心,上海200072 [2]同济大学口腔医学院·附属口腔医院儿童口腔科、上海牙组织修复与再生工程技术研究中心,上海200072

出　　处：《中华口腔医学杂志》2024年第7期706-714,共9页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81670962);科技部重点研发专项(2018YFE0202200);同济大学中央高校基本业务费专项资金(22120180196)。

摘　　要：目的探究转录因子激活蛋白2家族的成员转录因子激活蛋白2C(TFAP2C)在小鼠磨牙胚发育过程中的表达及其影响。方法实时荧光定量PCR(RT-qPCR)分析胚胎14.5(E14.5)天小鼠胚胎各器官及E12.5~E18.5天和出生后0~7(P0~P7)天的小鼠磨牙胚内Tfap2c的相对表达水平,冰冻切片免疫荧光染色显示Tfap2c在小鼠磨牙胚内的表达位置。采用牙胚体外培养的方法,探究敲降Tfap2c对小鼠磨牙胚发育的影响,RT-qPCR检测成牙本质细胞表达相关基因的相对表达水平。分离并提取小鼠牙胚间充质细胞进行培养,探究敲降Tfap2c对小鼠牙胚间充质细胞的影响,划痕愈合实验检测细胞迁移,细胞计数(CCK-8)法检测细胞增殖。结果Tfap2c基因在小鼠磨牙胚发育早期相对表达水平较高。E13.5天,Tfap2c在小鼠磨牙胚上皮组织与间充质组织内均有表达,二者的相对表达水平差异无统计学意义(t=1.06,P=0.472)。E14.5天,Tfap2c在小鼠磨牙胚初级釉结附近的间充质组织内表达,小鼠磨牙胚间充质组织内Tfap2c的相对表达水平(1.000±0.036)显著高于上皮组织(0.199±0.008)(t=37.29,P<0.001)。体外培养小鼠磨牙胚并敲降Tfap2c后,小鼠磨牙胚牙尖相对高度(0.708±0.171)及牙尖高度与牙冠高度的比值(0.321±0.068)均显著低于对照组(分别为1.000±0.287和0.483±0.166)(t=2.79,P=0.012;t=2.85,P=0.015);对照组的牙冠相对高度(1.078±0.206)及相对宽度(1.000±0.116)与敲降组的差异均无统计学意义(分别为0.993±0.254和0.999±0.122)(t=0.83,P=0.419;t=0.01,P=0.992)。体外培养小鼠磨牙胚内敲降Tfap2c,成牙本质细胞表达相关基因Dspp和Dmp1相对表达水平均显著降低(t=15.33,P<0.001;t=13.81,P<0.001)。在小鼠牙胚间充质细胞内敲降Tfap2c,划痕愈合实验显示,对照组细胞48 h划痕宽度显著小于敲降组(t=29.86,P=0.001);CCK-8法检测显示两组细胞间各时间点吸光度值的差异均无统计学意义(P>0.05);Tfap2c敲降组牙胚间充质细胞�Objective Explore the expression pattern of transcription factor activator protein 2C(TFAP2C)and identify the roles of Tfap2c during tooth development.Methods Real-time fluorescence quantitative PCR(RT-qPCR)was used to analyze the relative expression level of Tfap2c in various organs of embryonic day(E)14.5 mouse embryos and mouse molar germs at E12.5-E18.5 and postnatal day(P)0-P7.The expression position of Tfap2c in mouse molar germs was demonstrated by frozen section immunofluorescence staining.Cultured mandibular molar germs were transfected with control small interfering RNA(siRNA)or Tfap2c siRNA to evaluate the effect of Tfap2c on tooth molar germs development,and RT-qPCR was used to detect the relative expression level of genes related to odontoblast expression.Dental mesenchymal cells were isolated from E14.5 molar germs and transfected with control siRNA or Tfap2c siRNA,cell counting kit 8(CCK-8)and scratch healing test were applied to detect dental mesenchymal cell viability and migration.Results Tfap2c was highly expressed in the early development period of mouse molar germs.Tfap2c was expressed in the epithelial and mesenchymal tissues of E13.5 mouse molar germs and there was no significant difference of relative expression of Tfap2c between them(t=1.06,P=0.472).Tfap2c was expressed in mesenchymal tissues of E14.5 mouse molar germs and the relative expression of Tfap2c in mesenchymal tissues was significantly higher than epithelial tissues(t=37.29,P<0.0001).For molar germs transfected with Tfap2c siRNA,the relative height of cusps(0.708±0.171)and the ratio of cusp height and crown height(0.321±0.068)was significantly lower than control group(1.000±0.287 and 0.483±0.166)(t=2.79,P=0.012;t=2.85,P=0.015).But there was no significant difference in relative height(1.078±0.206,0.993±0.254,t=0.83,P=0.419)and relative width(1.000±0.116,0.999±0.122,t=0.01,P=0.992)of crowns between two groups.The relative expression level of genes related to odontoblast expression was decreased(Dspp:t=15.33,P<0.001;Dmp1:

关 键 词：转录因子 牙发育 转录因子激活蛋白2C 器官培养 成牙本质细胞分化 

分 类 号：R78[医药卫生—口腔医学]