【作者】 甘露；

【导师】 郑黎薇；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2022， 硕士

【摘要】 研究背景:牙釉质是覆盖于牙冠表面的矿化组织,具有美观,咀嚼和保护功能。发育过程中的信号干扰和突变极易导致牙釉质发育缺陷性疾病(Developmetntal defects of enamel,DDE),主要表现为牙釉质形态、色泽、结构和质地的改变,常对口腔正常生理功能造成不利影响,如牙釉质崩解、牙本质敏感、龋风险升高等。牙釉质形成有赖于关键信号通路WNT,FGF,TGFβ和BMP等的时空表达。表观遗传修饰调控扩展了我们对牙釉质发育机制的认识。N6-甲基化腺苷(m~6A)修饰是指腺苷的N6位发生甲基化,是真核细胞中最丰富的动态可逆的m RNA内部修饰,通过参与调控m RNA的选择性剪接、输出、衰变、稳定、翻译和定位,在RNA代谢和生理病理过程中起着重要作用,可调控昼夜节律、脂肪生成、精子发生、胚胎干细胞自我更新和分化、神经发育及胚胎发育等。但RNA m~6A甲基化修饰是否参与牙釉质形成调控仍然未知。研究目的:探究RNA m~6A甲基化及其相关修饰酶在小鼠牙釉质形成过程中的表达模式,为后期探索RNA m~6A甲基化在牙釉质形成中的调控作用提供基础研究基础。研究方法:本项目通过收集胚胎期E15.5(Embryonic period 15.5)、E18.5及出生后小鼠PN0(Postnatal day 0)、PN3、PN7不同发育时期小鼠颌骨样本,分离小鼠下颌第一磨牙牙胚成釉器,通过比色法检测牙上皮成釉向分化过程中m~6A甲基化的整体修饰水平,利用实时荧光定量PCR及Western Blot检测分析m~6A及其相关修饰酶的表达水平变化。进而利用免疫组织化学染色技术及免疫荧光染色技术检测m~6A及其相关修饰酶METTL3、FTO、YTHDC2在小鼠磨牙牙上皮成釉向分化及切牙上皮成釉向分化的时空表达情况。并应用m BMP4诱导小鼠切牙牙源性上皮干细胞(Dental epithelial stem cells,DESCs)成釉向分化模型,分析DESCs成釉向分化中的m~6A整体水平及METTL3、FTO、YTHDC2的表达水平。结果:在不同发育时期小鼠磨牙牙胚成釉器中,m~6A及m~6A甲基化相关修饰酶均可见表达。m~6A整体表达水平从E15.5到PN0表达水平逐渐升高,在PN0时期达到峰值。m~6A、METTL3表达水平从E15.5到PN0逐渐升高,PN3表达显著下降;FTO、YTHDC2表达水平从E15.5到PN3逐渐升高。在小鼠切牙牙上皮成釉向分化过程中,METTL3、FTO、YTHDC2在不同分化阶段的成釉细胞中均可见表达;应用m BMP4体外成功诱导小鼠切牙DESCs成釉向分化过程中,m~6A整体表达水平从诱导后0H到72H逐渐升高,在诱导后72H达峰值,诱导120H后表达下降;METTL3、FTO、YTHDC2表达水平逐渐升高。RNA m6A甲基化及其修饰酶在小鼠牙上皮中的差异性表达,表明METTL3、FTO、YTHDC2与牙上皮成釉向分化相关。结论:研究发现在小鼠磨牙、切牙牙上皮及DESCs成釉向分化进程中,m~6A甲基化整体表达水平及相关修饰酶METTL3、FTO、YTHDC2表达具有显著差异,提示m~6A及METTL3、FTO、YTHDC2与牙上皮成釉向分化相关,RNA m~6A甲基化可能参与调控牙釉质形成。更多还原

【Abstract】 Background:Enamel is a mineralized tissue covering the surface of tooth crown,which has aesthetic,chewable and protective functions.It is easy to Developmental defects of enamel(DDE)caused by signal interference and mutations during development,which are mainly manifested by changes in amelogenesis,color,structure and texture,and often adversely affect normal physiological functions of oral cavity.Such as enamel disintegration,dentin sensitivity,increased risk of caries,etc.Amelogenesis depends on the spatiotemporal expression of key signaling pathways such as WNT,FGF,TGFβand BMP.Epigenetic modification regulation has expanded our understanding of the mechanisms of amelogenesis.N6-methylated adenosine(m~6A)modification refers to the methylation of the N6 position of adenosine,which is the most abundant dynamic reversible internal modification of m RNA in eukaryotic cells.It plays an important role in RNA metabolism and physiological and pathological processes by participating in the regulation of selective splicing,output,decay,stability,translation and localization of m RNA.However,it is still unknown whether RNA m~6A methylation is involved in ameloblast differentiation of tooth epithelium during amelogenesis.Objective:To explore the expression pattern of RNA m~6A methylation and related modification enzymes in the process of tooth enamel formation in mice,providing a basic research basis for the later exploration of the regulatory role of RNA m6A methylation in amelogenesis.Methods:In this study,the mandible samples of mice at different developmental stages of E15.5,E18.5,PN0,PN3 and PN7 were collected,and the enamel ware of the tooth germ of the first mandibular molar of mice was separated,and the overall modification level of m6A methylation was detected by colorimetric method in the process of ameloblasts differentiation.The expression levels of m~6A methylation and related modification enzymes were analyzed by real-time fluorescence quantitative PCR and Western Blot.Immunohistochemical staining and immunofluorescence staining were used to detect the spatio-temporal expression of m~6A and its related modification enzymes METTL3,FTO and YTHDC2 in ameloblasts differentiation of mouse molar and incisor.The m~6A overall level and the expression of METTL3,FTO and YTHDC2 in the ameloblasts differentiation of the incisor of mice induced by m BMP4 were analyzed.Results:Both m~6A and m~6A methylation related modification enzymes were expressed in the enamel ware of mouse teeth in different developmental stages.The overall expression level of m~6A gradually increased from E15.5 to PN0,and reached the peak at PN0 stage.The expression levels of m~6A and METTL3 increased gradually from E15.5 to PN0,while the expression of PN3 decreased significantly.The expression levels of FTO and YTHDC2 increased gradually from E15.5 to PN3.METTL3,FTO and YTHDC2 were all expressed in ameloblasts at different stages of differentiation in the process of DESCs.Expression level of m~6A increased gradually from 0H to 72H after induction,peaked at 72H after induction,and decreased at 120H after inducted.The expression levels of METTL3,FTO and YTHDC2 increased gradually.The differential expression of RNA m~6A methylation and its modification enzyme in mouse dental epithelium indicated that METTL3,FTO and YTHDC2 were related to ameloblasts differentiation.Conclusion:It was found that the overall expression level of m~6A methylation and related modification enzymes METTL3,FTO and YTHDC2 were significantly different in the process of enamel differentiation of mouse molar and incisor epithelium and DESCs,indicating that m~6A,METTL3,FTO and YTHDC2 were related to enamel differentiation of dental epithelium.These results suggest that the epigenetic modification of RNA m~6A methylation may be involved in amelogenesis.更多还原