Runx2调控小鼠成牙本质细胞与成釉细胞的分化以及牙齿矿化的研究

发布时间：2018-06-02 22:07

本文选题：Runx2 + 转基因　；参考：《第四军医大学》2011年博士论文

【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。
[Abstract]:Runx2 is a transcription factor that plays an important role in osteoblast differentiation and tooth development. The specific matrix proteins in teeth and bone tissues are regulated by their transcriptional level. Runx2 deficiency leads to systemic hard tissue diseases including teeth, such as abnormal clavicle cranial bone dysplasia, CCDU. Runx2 gene knockout shows severe bone and tooth development disorders. Combined with the characteristics of transcription factors of Runx2 and its expression in tooth development, it can be inferred that Runx2 may also be an important transcriptional regulator for tooth development and mineralization. Recent studies have found that there is a spatiotemporal expression of Runx2 during tooth germ development, and it has been proved that Runx2 can regulate the expression of some specific matrix proteins in tooth development. At the same time, the Runx2 binding site was found in the promoter of amelogenin gene (-1641,92) and the promoter of dentin sialophosphorin gene (-3950 ~ 3106), through which Runx2 could significantly up-regulate the expression of these two genes. At present, it has been proved that Runx2 regulates the expression of a variety of mineralization-related proteins at the cellular level, but at the animal level, how does Runx2 regulate protein expression and what effect does it have on mineralization? Therefore, the mouse model of specific overexpression of Runx2 in ameloblast and odontoblast was established by transgenic method. To study the effect of Runx2 on the differentiation of ameloblasts and stromal cells and the phenotypic changes of enamel and dentin mineralization. Therefore, our project is divided into two parts: Part I Construction of transgenic mice The plasmid vectors of pBluescript II KS(-Amelogenin Promoter-Runx2 and pBluescript II KS(-DSPP Promoter-Runx2 were constructed respectively. By microinjection, the linearized plasmid was injected into the fertilized eggs of mice to construct transgenic mice, and specific primers were designed to identify the gene integration and expression of transgenic mice by using PCR technique. The high expression mouse lines were screened by qRT-PCR for phenotypic analysis. The second part: phenotypic Analysis of transgenic mice Phenotypic Analysis of DSPP Promoter-Runx2 transgenic mice In the initial stage of dentine formation, the odontoblast of transgenic mice completely lost its high columnar shape and polarization direction, the structure of dentin and dentine tubules disappeared in the Prophase, and the dentine became thinner and presented homogenous bone matrix structure. The new bone like structure appeared in the pulp cavity, which indicated that Runx2 inhibited the final differentiation and maturation of odontoblast and induced the differentiation of odontoblast into osteoblast. At the same time, the expression of DSPP and the formation of dentin were strongly inhibited, and osteoid matrix tissue was formed to replace the normal dentin. Phenotypic Analysis of Amelogenin Promoter-Runx2 transgenic mice in experiment 2 The results showed that the ameloblasts of transgenic mice lost their high columnar morphology and polarization, the formation of enamel matrix was inhibited, the ameloblast was blocked at the early stage of ameloblast secretion, and the expression of amelogenin was down-regulated. The results showed that Runx2 inhibited the expression of amelogenin and the differentiation of ameloblasts, caused the formation of enamel matrix, caused incomplete formation of enamel, and induced the metastasis and differentiation of ameloblast.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

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