本文選題：Runx2 + 轉(zhuǎn)基因　； 參考：《第四軍醫(yī)大學(xué)》2011年博士論文

【摘要】：Runx2是一種在成骨細(xì)胞分化及牙齒的發(fā)育過程中起著重要作用的轉(zhuǎn)錄因子,牙齒以及骨組織中的特異性基質(zhì)蛋白在轉(zhuǎn)錄水平均受其調(diào)控。Runx2缺陷導(dǎo)致包括牙齒在內(nèi)的全身硬組織疾病,如鎖骨顱骨發(fā)育異常(CCD)。Runx2基因敲除表現(xiàn)出嚴(yán)重的骨及牙齒發(fā)育障礙。結(jié)合Runx2的轉(zhuǎn)錄因子特性,以及在牙齒發(fā)育過程中的表達(dá)特征,可以推測Runx2可能也是牙齒發(fā)育和礦化的重要轉(zhuǎn)錄調(diào)控因子。最近有研究發(fā)現(xiàn),在牙胚發(fā)育過程中Runx2存在著表達(dá)的時(shí)空性,并且證實(shí)Runx2可以調(diào)控牙齒發(fā)育中的某些特異性基質(zhì)蛋白的表達(dá)。同時(shí),有結(jié)果顯示,釉原蛋白基因的啟動(dòng)子(-1641、+92)和牙本質(zhì)涎磷蛋白基因的啟動(dòng)子(-3950、-3106)上存在Runx2結(jié)合位點(diǎn), Runx2可以通過該位點(diǎn)顯著上調(diào)這兩種基因的表達(dá)。目前,在細(xì)胞水平上已經(jīng)證實(shí),Runx2調(diào)控著多種與礦化相關(guān)蛋白的表達(dá),但在動(dòng)物水平上,Runx2又是如何調(diào)控蛋白表達(dá)以及對礦化又有哪些影響呢? 因此,本課題通過轉(zhuǎn)基因手段分別建立在成釉細(xì)胞與成牙本質(zhì)細(xì)胞中特異性過表達(dá)Runx2的小鼠模型,研究Runx2對分化晚期的成釉細(xì)胞與成本質(zhì)細(xì)胞分化的影響以及因此造成的牙釉質(zhì)與牙本質(zhì)礦化的表型改變。為此,我們的課題共分為兩部分: 第一部分構(gòu)建轉(zhuǎn)基因小鼠 我們分別構(gòu)建pBluescript II KS(+)-Amelogenin Promoter-Runx2和pBluescript II KS(+)-DSPP Promoter-Runx2質(zhì)粒載體。通過顯微注射,將線性化的質(zhì)粒注入小鼠受精卵中,構(gòu)建轉(zhuǎn)基因小鼠,并且設(shè)計(jì)特異性引物,利用PCR技術(shù),鑒定轉(zhuǎn)基因小鼠的基因整合和表達(dá)情況。通過qRT-PCR篩選表達(dá)高的鼠系進(jìn)行表型的分析。 第二部分轉(zhuǎn)基因小鼠表型分析 實(shí)驗(yàn)一DSPP Promoter-Runx2轉(zhuǎn)基因小鼠表型分析 在牙本質(zhì)形成的啟始階段,轉(zhuǎn)基因小鼠成牙本質(zhì)細(xì)胞完全喪失本身的高柱狀形態(tài)和極化方向,前期牙本質(zhì)和牙本質(zhì)小管結(jié)構(gòu)消失,牙本質(zhì)變薄呈均質(zhì)狀的骨基質(zhì)樣結(jié)構(gòu),并且髓腔中出現(xiàn)新生的骨樣結(jié)構(gòu),表明Runx2抑制了成牙本質(zhì)細(xì)胞的最終分化成熟,并誘導(dǎo)處于分化晚期狀態(tài)的成牙本質(zhì)細(xì)胞向成骨細(xì)胞分化。同時(shí),強(qiáng)烈抑制DSPP的表達(dá)和牙本質(zhì)的形成,形成類骨基質(zhì)樣組織替代正常的牙本質(zhì)。 實(shí)驗(yàn)二Amelogenin Promoter-Runx2轉(zhuǎn)基因小鼠表型分析 結(jié)果發(fā)現(xiàn),轉(zhuǎn)基因小鼠成釉細(xì)胞失去原有的高柱狀形態(tài)和極化特征,釉基質(zhì)的形成受到抑制,釉基質(zhì)在成釉細(xì)胞分泌早期階段就已被阻斷,并且釉原蛋白表達(dá)下調(diào)明顯,表明Runx2抑制了釉原蛋白的表達(dá)和成釉細(xì)胞的分化,導(dǎo)致釉基質(zhì)形成障礙,造成釉質(zhì)形成不全,并誘導(dǎo)了成釉細(xì)胞的轉(zhuǎn)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R346

【參考文獻(xiàn)】

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1 顧新豐;蔣W，

本文編號：1970372