【作者】 张冰；

【导师】 徐欣； 文勇；

【作者基本信息】 山东大学 ， 口腔临床医学， 2016， 硕士

【摘要】 Yap (Yes-associated protein)是重要的转录共激活因子,位于Hippo信号通路下游,首次于1995年在果蝇体内发现。有研究报道Yap在多种组织器官中均存在表达,例肠、肝脏及神经管等。Taz (transcriptional coactivator with PDZ-binding motif)是一个14-3-3结合蛋白,含有一个保守的WW结构域。Taz为Hippo信号通路下游另一个重要的转录因子,是Yap的旁系同源蛋白。经典的Hippo信号通路控制组织器官的大小主要是通过调控细胞的增殖以及凋亡来实现。文献研究证实Yap/Taz在调控器官大小、干细胞自我更新、增殖及分化等方面都发挥作用。近年来有关Yap在牙胚早期发育及小鼠切牙发育中的相关报道,但对于Yap在大鼠磨牙发育过程中的研究尚未见相关报道,此外,Taz作为Yap的同源蛋白,是否参与牙齿的发育尚无研究,由此我们推测Yap/Taz可能在大鼠磨牙发育中具有特异性的表达。颅颌面骨与牙体组织的发育都源于神经嵴,在基因的表达上可能存在一定的相似性,颌骨是颅面部重要的解剖结构之一,研究发现Taz与Runx2相互作用可以提高Runx2的转录活性,促进成骨细胞的分化,在骨形成过程中发挥重要的作用,由此我们推测Taz与Runx2可能参与颌骨的修复再生过程。基于我们的推测,本研究分为两部分,第一部分以Sprague Dawley大鼠为实验动物,通过免疫组化染色及实时荧光定量PCR观察Yap/Taz在大鼠下颌第一磨牙发育过程中的时空特异性表达及检测Yap/Taz相对表达量的变化情况；第二部分构建Sprague Dawley大鼠颌骨缺损模型,通过免疫组化染色及蛋白免疫印迹检测Taz及Runx2在大鼠颌骨缺损再生过程中的表达变化情况,为探究Yap/Taz在牙齿发育及颌骨组织再生中的作用提供理论依据。第一部分Yap/Taz在大鼠下颌第一磨牙发育过程中的时空特异性表达目的：建立Sprague Dawley大鼠牙齿发育模型,观察Yap/Taz在下颌第一磨牙发育中的时空特异性表达。方法：10周龄Sprague Dawley大鼠雌雄1：1合笼,次日检栓,阴栓出现的当天中午即为受孕0.5天。获取胚胎14.5(E14.5)、16.5和18.5天及新出生(PN0)、出生后3、7、14、21和28天牙齿发育标本,通过常规固定、脱钙、脱水及石蜡包埋后,利用苏木素-伊红染色(HE染色)观察大鼠磨牙的发育形态及利用免疫组织化学染色观察Yap/Taz的时空特异性表达。此外,通过实时荧光定量PCR技术从基因水平检测Yap/Taz在出生后(PN0、PN4、PN7、PN10)大鼠牙齿发育中相对表达量的变化。结果：(1)免疫组织化学染色结果：在E14.5和E16.5天,Yap/Taz在口腔上皮和间充质中呈阳性表达,在E18.5天,Yap/Taz表达于牙乳头和整个成釉器。随着牙胚发育,在PN0、PN3、PN7天,免疫组化染色观察在成釉细胞、中间层及成牙本质细胞中均有Yap/Taz的表达,同时,在PN7天,阳性的Yap/Taz表达于上皮根鞘,在PN3、PN7、PN14天,未矿化的釉质基质中存在Yap的表达。在PN21及PN28天,Yap/Taz在尚存在分泌功能的成牙本质细胞中弱表达,然而Taz在血管内皮细胞及牙周膜和牙槽骨等牙周组织中表达较为明显。(2)实时荧光定量PCR结果：通过实时荧光定量PCR检测PN0、PN4、PN7及PN10天下颌第一磨牙牙胚中Yap/Taz相对表达量的变化情况。结果显示Yap与Taz有相似的表达趋势,PN0天,Yap/Taz的表达量相对较低,随着牙胚发育至PN4及PN7天,Yap/Taz表达显著升高,PN10天,Yap/Taz表达又出现下降趋势。PN4及PN7天,Yap/Taz的表达量相近,均明显高于PN0及PN10天。结论：(1) Yap/Taz在大鼠下颌第一磨牙发育过程中呈特异性表达与定位。(2) Yap/Taz可能参与成釉细胞及成牙本质细胞的分化,釉质的矿化、牙冠和牙根的形成以及牙周组织发育。第二部分Taz在大鼠颌骨缺损再生过程中的初步研究目的：构建Sprague Dawley大鼠颌骨缺损模型,初步探究Taz在颌骨缺损再生过程中的作用。方法：从山东大学实验动物中心购买8周龄雄性Sprague Dawley大鼠20只,于大鼠两侧下颌骨约第一、第二磨牙颊侧构建颌骨缺损模型,分别于术后3、7、14及21天取材,通过免疫组化染色观察Taz及Runx2在颌骨缺损再生过程中的表达与定位,通过蛋白免疫印迹(Western blotting)检测颌骨缺损再生过程蛋白表达水平的变化。结果：(1)免疫组织化学染色结果：在颌骨愈合的第3天,免疫组化染色显示生物膜处附着少量成纤维细胞存在Taz及Runx2的阳性表达,周围的肌纤维中可见明显的Taz及Runx2阳性着色,此外,在牙槽骨中的血管内皮细胞也存在明显阳性表达；第7天,Taz在迁移至肉芽组织中的成纤维细胞以及血管内皮细胞中呈阳性表达；第14天,骨小梁呈现不规则的排列,胶原纤维在新生骨组织处为编织状且排列无序。与早期愈合阶段相比,第21天新生骨组织面积明显变大,且骨质相对致密,免疫组化染色可见在14及21天毛细血管内皮细胞、类骨质样组织部位有Taz与Runx2的阳性表达,此外Taz在骨陷窝处可见明显的阳性表达。(2)蛋白免疫印迹结果：在大鼠颌骨缺损修复过程中,Taz及Runx2均有表达。以GAPDH为内参,将Taz与Runx2进行标准化分析发现随着愈合时间延长,二者的表达量呈现相对升高的趋势。结论：(1)Taz及Runx2在大鼠颌骨缺损再生过程中呈特异性表达。(2)Taz及Runx2可能在大鼠颌骨缺损再生过程中发挥重要的调控作用。更多还原

【Abstract】 Yap (Yes-associated protein) as an effective downstream transcription factors of the Hippo signaling pathway, which was discovered in Drosophila in 1995. Previous studies demonstrated that Yap was expressed in a variety of tissues and organs, such as intestine, liver and neural tube etc. Taz (transcriptional coactivator with PDZ-binding motif) is a 14-3-3 binding protein, containing a conserved WW domain. Taz is another important transcription factor in Hippo signaling pathway and is homologous with Yap. Hippo signaling pathway can manage organ size through regulating cell proliferation and apoptosis. Research has confirmed that Yap/Taz played an important role in regulating organ size and stem cells self-renewal, proliferation and differentiation. There are relevant reports about Yap in the development of early tooth germ and mouse incisor. However, the research in rat molar remains elusive. In addition, it has not been reported that Taz as a homologous of Yap was involved in tooth development. We hypothesized that Yap/Taz may be involved in the process of molar development. The development of cranio maxillofacial bone and tooth are derived from the neural crest and the gene expression may have some similarities. The jaw is one of the important anatomic structures in craniofacial bone. TAZ can interact with Runx2, and then enhance the transcriptional activity of Runx2. What’s more, it can promote the differentiation of ostoblasts and the formation of bone tissue. What we speculated that Taz and Runx2 may take part in the jaw repair and regeneration. This study was divided into two parts and we used Sprague Dawley rats as the experimental animals and detected the spatial-temporal expression and the relative expression level of Yap/Taz through immunohistochemistry and qRTPCR in the first part. The second part, we constructed model of jaw tissue defect and tested the expression of Taz and Runx2 through immunohistochemistry and Western blotting. Our study may provide corresponding experimental basis for researching the role of Yap/Taz in tooth development and jaw tissue regeneration.Part I Expression and localization of Yap and Taz during the development of mandibular first molar in ratsObjective:The purpose of this study was to observe the spatial-temporal expression pattern of Yap/Taz through establishing the experimental animal model of tooth development in Sprague Dawley rats.Methods:10w Sprague Dawley rats (male and female with1:1) were mated to obtain tissue samples of embryonic and postnatal rats. Noon of the day on which the vaginal plug was found was regarded as embryonic day 0.5 (E0.5). Tissue samples include embryonic rat heads (E14.5, E16.5, E18.5) and postnatal rat mandibles from new birth to postnatal 4 weeks (PNO, PN3, PN7, PN14, PN21, PN28). All samples were treated with conventional fixed, decalcified, dehydration and paraffin embedded. Hematoxylin-eosin staining (HE staining) was used to observe the development morphology of mandibular first molar in rats and immunohistochemistry was used to observe the spatial-temporal expression and localization of Yap/Taz. In addition, qRTPCR was used to validate the relative expression of Yap/Taz gene at PNO, PN4, PN7 and PN10.Results:(1) The results of immunohistochemistry staining:Yap/Taz were found in oral epithelium and mesenchyme at E14.5 and E16.5.At E18.5, Yap/Taz was detected in dental papilla and whole enamel organ. At PNO, PN3 and PN7, positive expression existed in ameloblasts, odontoblasts and stratum intermedium. Meanwhlie, Yap/Taz were expressed in Hertwig’s epithelial root sheath (HERS) distinctly at PN7. At PN3, PN7 and PN14, Yap was detected in enamel matrix. From PN21 to PN28, parts of odontoblast which owned secretory function still remained Yap/Taz expression weakly during this period. However, Taz was highly detected in endothelial cells of blood vessels, periodontal ligament and alveolar bone.(2) The results of qRTPCR:To assess the Yap/Taz gene expression during the development of mandibular first molar in rats, we performed qRTPCR analyses in tooth germ at PNO, PN4, PN7 and PN10. The result of qRTPCR is that the expression of Yap/Taz in tooth germ of PNO was low. The level of Yap/Taz expression at PN4 and PN7 increased significantly than PN10. The level of Yap/Taz expression at PN4 was similar to PN7 and they were higher than PN0 and PN10.Conclusion:(1) Yap/Taz appeared spatial-temporal expression during the development of mandibular first molar in rats.(2) Yap/Taz might be involved in differentiation of ameloblasts and odontoblasts, enamel mineralization, crown morphogenesis, root formation and periodontium development.Part Ⅱ Preliminary study of Taz during the process of jaw regeneration in rats Objective:The purpose of this study was to construct the experimental animal model of jaw tissue damage in Sprague Dawley rats and to explore the role of Taz in the healing process of jaw tissues in rats.Methods:We pruchased 20 male Sprague Dawley rats (8 weeks) from Laboratory Animal Center of Shandong University. We constructed jaw defect model on the first and second molar buccal of mandible in rats. Samples were obtained from 3 day,7 day,14 day and 21 day after operation respectively. The expression and localization of Taz and Runx2 during the process of tissue wound healing was observed through immunohistochemical staining. Western blotting was used to detect the protein expression level during the process of damage healing.Results:(1)The results of immunohistochemistry staining:On the third days of jaw tissue defect healing, immunohistochemical staining showed the positive expression of Taz and Runx2 in fibroblasts attached to biological membranes and positive staining in the muscle fiber was obvious. In addition, the endothelial cells of blood in alveolar bone was Taz positive expression. The seventh days in tissue healing, the expression of Taz and Runx2 in endothelial cells and fibroblasts was positive. On the fourteenth days of healing, bone cell in the. new bone was not mature, irregular arrangement and lack of canalicular system. What’s more, the collagen fibers were disordered. On the 21st day of the healing tissue, bone tissue area which was newly formed was significantly larger than before, and bone mineral density was increase. Taz and Runx2 expression were detected in endothelial cells of blood and osteoid tissue. In addition, the positive expression of Taz was detected in bone lacuna.(2) Western blotting results:Taz and Runx2 were expressed during the wound healing of jaw. With GAPDH as an internal reference, TAZ and Runx2 were standardized analysis. The expression of Taz and Runx2 were gradually increased during the wound healing.Conclusions:(1) Taz and Runx2 were specific expression during the wound healing of jaw in rats.(2) Taz and Runx2 might play an important role in the regeneration of jaw in rats.更多还原