利用BMP基因敲除小鼠模型研究BMP信号通路导致釉质生成不良的分子机制

Matrix proteinases play important roles in the mineralization of enamel. Their deficiencies result in Amelogenesis Imperfecta, impairing tooth function and aesthetic appearance. Currently, few studies on the molecular mechanism of matrix proteinase expression in ameloblasts cause the absence of early diagnosis and treatment on Amelogenesis Imperfecta. Our preliminary work found that the specific ablation of BMP signal molecules in dental epithelium did not affecte differentiation of ameloblasts, but still caused Amelogenesis Imperfecta, which suggested that BMP signaling pathways promoted enamel mineralization by regulating the expression and activities of matrix proteinases. First, we plan to explore the regulatory mechanism of BMP signal molecules on ameloblast differentiation and the expression of matrix proteins, especially the expression of matrix proteinases by exploiting epithelial deletion of BMP2 and BMP4 mice model and ameloblast cell line；Secondly, we are going to identify the special BMP signaling pathway controlling matrix proteinase expression by supplementing small molecular inhibitors to BMP signaling pathways; Finally, the transgene and small molecular activators of BMP signaling pathway will be employed to rescue the defects of enamel mineralization. Our study will systematically explicate the network of BMP signaling pathway regulating enamel mineralization and provide new methodology and strategy to the clinic diagnosis and treatment on Amelogenesis Imperfecta.

基质蛋白酶在牙釉质矿化中起重要作用，其缺陷会导致人釉质生成不良，严重影响牙齿功能和外观审美。目前对于调控成釉质细胞中基质蛋白酶表达的分子机制研究较少，因而缺乏对釉质生成不良的早期诊断和防治的手段。我们发现在牙上皮内特异性敲除BMP信号分子，成釉细胞分化正常，但却会导致小鼠釉质生成不良。这提示BMP信号通路很可能通过调控基质蛋白酶的表达和活性来影响牙釉质矿化。本研究拟利用上皮特异性BMP2和BMP4敲除小鼠模型和成釉质细胞系，明确BMP信号蛋白对于成釉质细胞分化和基质蛋白，尤其是基质蛋白酶表达的调控机制；继而通过添加特定BMP信号通路抑制剂，鉴定出控制基质蛋白酶表达的具体信号通路；最后将利用转基因或添加信号通路激活剂的方法，尝试挽救BMP缺陷小鼠的釉质矿化不良。本研究有望系统阐明BMP信号通路调控牙釉质矿化的分子网路，为临床早期诊断和治疗牙釉质生成不良开辟新的方法和策略。

骨形态发生蛋白（BMPs）作为一类重要的生长因子，其在早期牙发育过程中的作用已经进行了大量的研究，但其对成釉细胞分化成熟及牙釉质形成的确切作用尚不明确。课题组前期工作发现BMP2和BMP4上皮内条件性敲除可以导致小鼠牙釉质生成不良（Amelogenesis Imperfecta，AI）。在体内研究中，BMP2和BMP4的缺失影响BMP信号通路，对牙釉质的发育、形成以及釉质基质蛋白和釉基质蛋白酶的变化产生了影响。体外研究中，建立牙胚器官模型，应用BMP2和BMP4重组蛋白以及BMP信号通路特异性抑制剂分别激活和抑制BMP信号通路，观察BMP2和BMP4在体外对BMP信号通路以及釉质发育相关基因的影响。为了进一步验证BMP经典信号通路的作用，本实验又建立了Smad4上皮内条件性基因敲除小鼠模型，证明了经典BMP信号通路在釉质生成中的重要作用。综上，课题组认为BMP2和BMP4基因敲除主要是通过经典信号通路（BMP/Smad4）影响牙釉质的生成；BMP2和BMP4基因缺失影响了釉质基质蛋白和蛋白酶基因的表达；经典的BMP/Smad4通路和非经典BMP/p38通路对釉质的发育都有重要的影响。