Furin在牙本质形成以及相关疾病中作用的实验研究

Dentin extracellular matrix proteins, predominantly expressed in odontoblasts and secreted into dentin matrix, are believed to play key biological roles in dentinogenesis and related dental diseases. Post-translational modifications are essential for their activation; however, the underlying activation mechanism remains unknown. .Our previous studies have indicated that Matrix Extracellular Phosphoglycoprotein (MEPE) is cleaved by furin (a typical proprotein convertase) into two fragments, C-terminal fragment and N-terminal fragment, during its secretion by odontoblasts, and Dentin Sialophosphoprotein (DSPP) is also the substrate of furin. We proposed the activation model of DSPP catalyzed by furin in dentinogenesis, and the hypothesis that this proteolytic processing may play important roles in the defective mineralization of dentin..In this study, first we will establishes furin over-expression and knock down odontoblasts cell line by gene transfection and RNAi respectively, to determine the proteolytic processing of DSPP and dentinogenesis in vitro. Then odontoblasts specific conditioned furin knock out mouse will be developed by Cre/loxP system, to determine the proteolytic processing of DSPP and dentinogenesis in vivo. Last, we will explore the possible mechanism of defective mineralization of dentin in familial DGI and DD etc., based on the analysis of DSPP cleavage of dentin in these patients. .Our study will clarify the mechanism of DSPP activation via proteolytic processing, uncover the importance of post-translational modifications of dentin extracellular matrix proteins in dentinogenesis, and probe into the pathogenesis of hypoplastic dentinogenesis, which is much helpful for the prevention and cure of such diseases.

牙本质基质蛋白的裂解活化在牙本质形成中具有重要作用，发生异常导致矿化不良，至今机理尚不明确。我们的研究发现：前蛋白转化酶furin裂解活化牙本质基质蛋白MEPE；牙本质中主要的基质蛋白DSPP也是furin的底物。我们提出假说：牙本质形成中，furin裂解活化DSPP形成DSP和DPP，调控牙本质基质形成和矿化。本研究首先构建furin过表达和基因敲除的成牙本质细胞系，分析DSPP裂解活化和体外牙本质形成情况；然后利用Cre/loxP系统，构建成牙本质细胞特异性的furin基因敲除小鼠，分析体内DSPP裂解活化情况和牙本质表型，包括牙本质生长速度、力学性能、组织学和基质蛋白情况等；最后收集牙本质发育性疾病患牙，检测furin活性和DSPP裂解活化情况。本课题将阐明furin裂解活化DSPP过程，明确基质蛋白翻译后修饰在牙本质形成中作用，揭示牙本质发育性疾病的可能致病机理，为其防治提供思路。

牙本质形成是牙齿发育的重要阶段，也是牙本质牙髓复合体损伤修复过程中的关键环节。成牙本质细胞分化成熟后，分泌细胞外基质蛋白形成牙本质基质，调控羟基磷灰石晶体的成核和聚集，牙本质矿化形成。本项目提出DSPP裂解活化模型，即furin在成牙本质细胞内裂解DSPP形成DSP和DPP，调控牙本质细胞外基质的形成，影响牙本质矿化过程，是牙本质发育不全的可能致病途径。本研究从牙本质细胞外基质蛋白翻译后修饰的角度探讨牙本质形成的机制，为疾病的防治提供思路。.1. furin裂解DSPP蛋白质形成DSP和DPP。将外源性DSPP转染furin基因缺陷性人结肠癌细胞系LoVo细胞中，仅有全长DSPP蛋白质表达。体外酶解实验证实furin可以裂解DSPP蛋白质形成DSP和DPP。.2. 体外furin 裂解活化DSPP，调控牙本质形成。构建furin基因过表达稳定细胞系OLC-furin和基因沉默的稳定细胞系OLC-furin-miR。分析DSPP裂解和牙本质形成情况，结果显示：OLC-furin中DSPP的裂解情况与对照组OLC无明显差异，无DSPP全长，具有相似的矿化能力；OLC-furin-miR中则基本为DSPP全长，仅有少量DSP片段，同时矿化能力较低。表明furin是DSPP裂解活化的重要蛋白酶，在成牙本质细胞内裂解DSPP形成DSP和DPP，并分泌至细胞外。.3. 体内furin 裂解活化DSPP，调控牙本质形成。进口Furin KO-first小鼠胚胎以及DSPP-Cre活体小鼠，Furin KO-first小鼠胚胎杂交Flp工具鼠后即为Furin CKO小鼠，再与DSPP-Cre小鼠杂交筛选获得子代furin（-/-）Cre（+），即为牙本质细胞特异性furin条件性基因敲除小鼠。表型分析结果：CKO小鼠出生后3天、7天、14天磨牙牙胚中存在DSPP全长蛋白，提示DSPP未完全裂解；牙本质层比野生型薄，与成牙本质细胞之间有间隙，牙本质小管结构不完整，成牙本质细胞排列方向散乱。出生后21天、28天的CKO小鼠与对照组相比无明显差异。.4. Furin在遗传性牙本质发育不良的可能致病机理。收集遗传性牙本质发育不良患者的脱落乳牙以及正畸原因拔除的恒牙，收集牙本质基质蛋白，与正常牙齿比较，Western blot显示发现患牙牙本质中存在DSPP全长蛋白，提示DSPP未完全裂解。