【作者】 李适廷；

【导师】 倪龙兴； 朱庆林；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2011， 博士

【摘要】 Runx2是一种在成骨细胞分化及牙齿的发育过程中起着重要作用的转录因子,牙齿以及骨组织中的特异性基质蛋白在转录水平均受其调控。Runx2缺陷导致包括牙齿在内的全身硬组织疾病,如锁骨颅骨发育异常(CCD)。Runx2基因敲除表现出严重的骨及牙齿发育障碍。结合Runx2的转录因子特性,以及在牙齿发育过程中的表达特征,可以推测Runx2可能也是牙齿发育和矿化的重要转录调控因子。最近有研究发现,在牙胚发育过程中Runx2存在着表达的时空性,并且证实Runx2可以调控牙齿发育中的某些特异性基质蛋白的表达。同时,有结果显示,釉原蛋白基因的启动子(-1641、+92)和牙本质涎磷蛋白基因的启动子(-3950、-3106)上存在Runx2结合位点, Runx2可以通过该位点显著上调这两种基因的表达。目前,在细胞水平上已经证实,Runx2调控着多种与矿化相关蛋白的表达,但在动物水平上,Runx2又是如何调控蛋白表达以及对矿化又有哪些影响呢?因此,本课题通过转基因手段分别建立在成釉细胞与成牙本质细胞中特异性过表达Runx2的小鼠模型,研究Runx2对分化晚期的成釉细胞与成本质细胞分化的影响以及因此造成的牙釉质与牙本质矿化的表型改变。为此,我们的课题共分为两部分:第一部分构建转基因小鼠我们分别构建pBluescript II KS(+)-Amelogenin Promoter-Runx2和pBluescript II KS(+)-DSPP Promoter-Runx2质粒载体。通过显微注射,将线性化的质粒注入小鼠受精卵中,构建转基因小鼠,并且设计特异性引物,利用PCR技术,鉴定转基因小鼠的基因整合和表达情况。通过qRT-PCR筛选表达高的鼠系进行表型的分析。第二部分转基因小鼠表型分析实验一DSPP Promoter-Runx2转基因小鼠表型分析在牙本质形成的启始阶段,转基因小鼠成牙本质细胞完全丧失本身的高柱状形态和极化方向,前期牙本质和牙本质小管结构消失,牙本质变薄呈均质状的骨基质样结构,并且髓腔中出现新生的骨样结构,表明Runx2抑制了成牙本质细胞的最终分化成熟,并诱导处于分化晚期状态的成牙本质细胞向成骨细胞分化。同时,强烈抑制DSPP的表达和牙本质的形成,形成类骨基质样组织替代正常的牙本质。实验二Amelogenin Promoter-Runx2转基因小鼠表型分析结果发现,转基因小鼠成釉细胞失去原有的高柱状形态和极化特征,釉基质的形成受到抑制,釉基质在成釉细胞分泌早期阶段就已被阻断,并且釉原蛋白表达下调明显,表明Runx2抑制了釉原蛋白的表达和成釉细胞的分化,导致釉基质形成障碍,造成釉质形成不全,并诱导了成釉细胞的转移分化。更多还原

【Abstract】 Runx2 is an important transcription factor in osteoblast differentiation and dental development. Most of the specific matrix proteins expressed in tooth and bone were regulated by Runx2 at transcriptional level. Runx2 gene deficiency results in Systemic hard tissue diseases including tooth, for example: cleidocranial dysplasia (CCD). Severe aberration of bone and dental development appeared in Runx2 gene knockout mice. Combining transcription characteristics of Runx2 with its expression feature in tooth development, we think Runx2 may be one of the most important transcription factor in tooth mineralization and development. Latest research shows that Runx2 expressed temprally and spacially in tooth germ development and proves that Runx2 can regulate the expression of some specific matrix protein in tooth development. Meanwhile, finding reveals that there are binding sites for Runx2 at Amelogenin promotor(-1641、+92)and DSPP promotor(-3950、-3106), and Runx2 can up-ragulate the expression of the two proteins by this binding sites. Recently, there is proof that many matrix proteins related to mineralization are regulated by Runx2 at cellular level. But we do not know what effection and function of Runx2 would be in animals?Thus, in this study, two transgenic mice were generated, in which Runx2 was over expressed in ameloblast or odontoblast respectively. Next, the differentiation of ameloblast as well as odontalblast was studied and the mineralization of enamel and dentin were characterized. So our studies includedtwo parts:Part one: Construction of transgenic miceWe generated pBluescript II KS(+)-Amelogenin Promoter-Runx2 and pBluescript II KS(+)-DSPP Promoter-Runx2 plasmids, respectively. Then we generated transgenic mice by microinjecting linearized plasmids into oosperm, and designed the primers to identify the gene integration and overexpression of the transgene by PCR. We selected highly expressed lines to analyze the phenotype of the transgenic mice.Part two: Phenotypic anylasis of transgenic mice1. Phenotypic anylasis of DSPP Promoter-Runx2 transgenic mice During the initiation and formation of dentin, the odontoblasts lost their tall columnar shape and polarization gradually. The structure of predentin and dentin tubuleswere lost and the osteoblast-like cells appeared, dentin became thinner, exhibitting homogeneous bone matrix-like structure. Some bone-like tissuesgrew into dental pulp chamber.These findings indicated Runx2 inhibited the terminal differentiation and maturationof odontoblast andinduced the transdifferentiation from odontoblast to osteoblast.Meanwhile, the DSPP expression level in dentin and dentin formation were dramatically reduced by Runx2, and Runx2 forced the transdifferentiated odontoblasts to secrete osteo-matrix instead of normal dentin matrix.2. Phenotypic anylasis of Amelogenin Promoter-Runx2 transgenic miceDuring the initiation and formation of enamel, ameloblasts lost their tall columnar shape and polarization gradually. Enamel matrix formation was dramatically inhibited.The formation of enamel matrix was blocked at the very beginning of the enamel matrix secreting stage, and the expression of amelogenin was down-regulated obviously. Conclusively, Runx2 induced the transdifferentiation of ameloblast and inhibited the expression of amelogenin and ameloblast differentiation, which resulted in enamel matrix lost and amelogenesis imperfecta,更多还原