母体糖尿病通过氧化应激调控Runx2基因表达影响牙囊发育的机制研究

Recent observations suggest that maternal diabetes during pregnancy may be link to the offspring's abnormality of tooth development. Previous studies indicated that change of stem cell niche resulted from oxidative stress induced by diabetes was involved in the abnormality of tooth development. Dental follicle stem cell, the mesenchymal stem cells derived from the neural crest, have stem cell properties and can be cultured as stem cells in vitro and differentiated in vivo into odontoblasts, cementoblasts and periodontal ligament cells,have an critical role during the development of tooth. Unfortunately, to date, the regulation of gene expression in response to dental follicle development has not been determined completely.Runt-related transcription factor 2 (RUNX2), also known as core-binding factor subunit alpha-1 (CBF-alpha-1), is a key transcription factor associated with dental follicle stem cell differentiation, recruitment of monocytes and osteoclastogenesis and the impairment of RUNX2 expression may be related to oxidative stress. However, the mechanism(s) whereby oxidative stress induced by pregnancy diabetes affects dental follicle stem cell RUNX2 expression and tooth development remain poorly understood. Here, we investigated in mice the effect of pregnancy diabetes on the development of fetal tooth in vivo and in vitro. Specifically, in aim 1,we produce the pregnancy diabetes female mice model by high-fat-diet and streptozotocin. The oxidative stress of dental follicle during the tooth development will be detected. And dental follicle stem cells proliferation, differentiation and apoptosis and osteoclastogenesis will also be detected by immunofluorescent staining, in situ hybridization and TRAP Staining. Aim 2 will investigate the affect of oxidative stress on the primary dental follicle stem cell proliferation, differentiation and apoptosis and the expression of RUNX2 signaling pathway. Findings from these experiments will quantify regulatory relationships among maternal diabetes, oxidative stress and RUNX2 signaling pathway in dental follicle stem cell. This effort will identify new molecular targets for therapeutic regimen to both prevent and treat abnormality of tooth development.

母体妊娠糖尿病可引起子代牙发育异常,其机制与糖尿病诱发的氧化应激微环境有关。牙囊干细胞是牙囊组织中具有多向分化潜能的间充质干细胞,增殖分化后形成牙周组织,在牙发育中具有重要作用。RUNX2是牙囊干细胞重要的调控因子,与牙囊干细胞分化、单核细胞募集和破骨形成有关,其表达的调控机制涉及氧化应激。然而,目前就妊娠糖尿病导致的氧化应激微环境对牙囊干细胞RUNX2表达及牙囊发育的影响尚不清楚。因此，本项目拟1）建立妊娠糖尿病小鼠模型，研究其对子代小鼠牙囊微环境氧化应激水平、牙囊细胞增殖、分化和RUNX2表达及单核细胞募集和破骨形成的影响;2）体外培养牙囊干细胞,研究氧化应激对RUNX2表达及牙囊干细胞生物学特性的影响和作用机制。本项目系统研究糖尿病导致的氧化应激微环境对牙囊干细胞RUNX2表达及其下游信号的影响和作用，揭示妊娠糖尿病导致牙发育异常的机理,为预防和治疗相关疾病提供理论依据和治疗靶点。

随着生活方式、饮食习惯的改变和婚育年龄的推迟，妊娠糖尿病的发病率逐年增高。妊娠糖尿病可导致子代多个组织或器官的发育异常。然而目前就妊娠糖尿病对牙齿发育的影响及其调控机制尚未见有系统的研究。本研究通过构建妊娠糖尿病大鼠模型，获取不同发育阶段的子代牙胚/牙齿，通过组织切片、免疫组织化学、TUNEL等方法研究妊娠糖尿病对牙胚发育过程中的细胞的增殖、分化和凋亡的影响；通过micro CT扫描及三维重建后定量分析，研究妊娠糖尿病对子代牙齿形态结构的影响；体外培养牙间充质细胞（牙乳头细胞、牙囊细胞）和牙上皮干细胞（切牙颈环上皮干细胞，Dental epithelial stem cell,DESC），通过基因敲减（RNAi）或过表达等方法研究妊娠糖尿病对子代牙胚细胞影响的调控机制；结果表明：1）妊娠糖尿病可显著抑制其子代牙胚细胞增殖、促进细胞凋亡，抑制细胞的分化；2）妊娠糖尿病激活子代牙胚细胞TLR4/NF-kappa B信号通路，并通过TLR4/NF-kappa B信号通路调控子代牙胚细胞的增殖、凋亡和分化；3）研究证实妊娠糖尿病抑制子代切牙颈环细胞APEX1表达，APEX1通过ERK和JNK信号通路调控DNMT1的表达，从而影响切牙颈环细胞DNA甲基化水平;4）妊娠糖尿病显著上调子代牙囊细胞Runx2基因表达，体外过表达Runx2基因可促进单核细胞向破骨细胞分化相关基因的表达显著上调。本研究首次系统研究妊娠糖尿病对子代牙齿发育的影响，本研究结果对于探究牙发育的调控机制具有重要的理论和实际意义，也为临床上牙发育异常的诊断和治疗、组织工程牙齿再生提供理论和实验依据。