【作者】 沈永岱；

【导师】 田卫东；

【作者基本信息】 四川大学 ， 口腔临床医学， 2007， 博士

【摘要】 哺乳动物牙齿发育需要经过较长时间。在这一系列由牙齿发生到进一步发育的过程中，从口腔上皮增厚期到牙板期、蕾状期、帽状期、钟状期和细胞终末分化期等每个时期均具有明确的发育特征。由外胚层来源的牙釉上皮细胞及颅神经嵴来源的间充质细胞在从定向分化到牙发生发育过程中，均要接受来自于自分泌或者旁分泌的分子信号转导通路的调控。其中重要的分子如音猬因子Shh(sonic hedgehog，Shh)，它是一种重要的分子信号蛋白质。由于果蝇的该基因突变会导致其幼虫体表出现许多刺突，状似刺猬，故称Hedgehog，它在脊椎动物中的同源基因为Shh，参与脊椎动物发育过程中极其复杂的信号转导过程，它参与软骨细胞分化、毛囊发育以及肢体形成等；wnt(wingless，wnt)是调控细胞生长增殖的关键信号转导通路，在胚胎正常发育和肿瘤发生中起着重要作用。同时，Shh、wnt信号转导通路在牙发生发育的过程中具有重要的作用。而slimb基因及其编码的蛋白质β传导素重复蛋白(βtransducin repeat-containing proteins，β-TrCP)，在果蝇及小鼠体内发挥对分子信号蛋白的降解作用，参与调节Shh、wnt信号转导通路，进而调节器官的正常发育。但是在牙发育过程中这些重要信号转导通路受slimb调控的生物学过程以及机制并不清楚。本研究就牙发育过程中Shh、wnt等信号转导通路中的关键分子在β传导素重复蛋白β-TrCP作用后，对其影响牙发育的作用机制进行了系统深入地研究。本研究旨在探索β-TrCP蛋白对牙发育的影响，以及slimb基因调节shh、wnt分子信号转导通路的作用，并研究其对形成牙齿的两种特异性细胞—牙釉上皮细胞、牙间充质细胞的作用，为进一步揭示牙发生发育的分子机制和牙再生研究提供理论依据。1．本研究以不同发育时期的小鼠牙胚为研究对象，取不同发育时期的牙胚标本，采用免疫组织化学LsAB法及RT-PCR法，从蛋白质和基因水平分别观察slimb／β-TrCP在牙胚不同发育阶段的表达特点。2．以体外培养的牙胚细胞为研究对象，采用牙胚细胞共同培养方法，用siRNA-slimb分别对牙釉上皮细胞、牙间充质细胞进行转染，然后采用RT-PCR法与western blot法检测siRNA-slimb的转染效率。在RNA干扰slimb沉默表达有效后，分别观察slimb基因沉默对这两种牙胚细胞生物学特性的影响。3．采用RT-PCR法观察slimb基因沉默后，牙釉上皮细胞与牙间充质细胞中牙发育相关基因的表达变化，以及Shh信号转导通路、wnt信号转导通路的基因表达变化。4．在胚胎发育不同时期的孕小鼠体内注射四种位点特异的siRNA-slimb的混和物，观察slimb基因有效沉默后，在不同时间点对小鼠牙胚发育的影响。实验结果显示：1．β-TrCP在牙胚发育不同时期呈现特征性表达，分别在胚胎E10.5的牙发生部位上皮与口腔上皮表达，在E13.5的牙蕾与邻接间充质组织中表达，以及在此后牙胚发育各期的牙上皮来源的细胞与间充质来源的细胞均有表达。在出生后的1d、3d、6d成釉细胞层与成牙本质细胞层胞浆中表达。与Shh、wnt两种分子信号转导通路的表达部位部分重叠。β-TrCP在胚胎发育时期组织中广泛表达，包括牙胚、毛囊、眼、皮肤以及黏膜等组织中表达。2．slimb负向调节Shh、wnt信号传导通路中的关键节点基因如GLI1、GLI3和β-catenin，从而参与调节牙发育。3．siRNA-slimb对牙胚组织中的牙釉上皮细胞作用研究显示，在slimb基因下调表达48h后，牙釉上皮细胞较对照组细胞增殖活性增加，细胞周期改变，出现较多量的未分化牙釉上皮细胞。4．siRNA-slimb对牙胚组织中的牙间充质细胞作用研究显示，在slimb基因下调表达后48h后，牙间充质细胞增殖活性增加，同时牙发育关键分子信号转导通路的基因表达发生改变。5．研究显示在牙釉上皮细胞、牙间充质细胞中转染slimb基因后，引起GLI1表达下调，从而阻碍了Shh信号转导通路，实质上干扰了Shh信号转导通路的正确传递。同时，slimb转基因后，分别引起牙釉上皮细胞、牙间充质细胞的增殖能力减弱，该基因超表达不引起shh、wnt10a基因下调表达，因此，slimb在负向调节Shh、wnt信号转导通路的同时，可能还有其它作用机制参与。6．在体内注射siRNA-slimb混和物后，在不同时间点观察其对小鼠胚胎中牙胚的影响。发现在E10.5和E13.5，在小鼠体内注射siRNA-slimb后，会引起胎鼠死亡；却对于发育中晚期的小鼠牙胚并未有明显影响，对已分化为成釉细胞与成牙本质细胞的牙源性细胞影响较小。综上所述，siRNA-slimb对体外培养的牙胚细胞增殖、分化等有一定的影响，它促进牙胚细胞的增殖，并将牙胚细胞维持在未分化状态。它对牙胚细胞周期产生明显影响，使牙胚细胞S期比例上升，DNA合成增加。在牙胚细胞中转基因研究显示，它可引起Shh、wnt信号转导通路中GLI1表达变化。因此，slimb是牙发育的关键基因之一。更多还原

【Abstract】 BACKGROUND: Along with the evolution and life lengthening of mankind, tooth loss may be the important factor of low life quality. Although modern dentistry technique could provide excellent quality of prosthetic for tooth loss, such as various kinds of denture, dental implants, however, it can not prevent tooth loss due to the little knowledge of mechanisms of tooth development. Tooth development may take a very sophisticated process and background. So, the study of mechanism is very important. The previous study had acquired lots of results; Tooth morphogenesis is regulated by epithelial-mesenchymal interactions and shares a similar molecular signaling network with other epithelial appendages. In tooth, the epithelial signaling centers function in three stages of morphogenesis. The initiation of tooth germ, the formation of crown base and the formation of each cusp. And there may be lots of signaling molecular constructing network participating in this process. It is well established that Among these signaling molecular, Sonic hedgehog (SHH) , and wingless signaling transduction pathway are very important to tooth morphogenesis and tooth development. The Hedgehog (Hh) signaling pathway controls many key developmental processes during animal embryogenesis. The fundamental importance of SHH in organogenesis was demonstrated of its role in patterning the brain, spinal cord, axial skeleton, limbs and epithelial mesenchymal interactions. Epithelial organs such as teeth are defined as those organs in which epithelial-derived cells become the major component of the organ following epithelial-mesenchymal interactions. And this is the main obstacle for tooth regeneration in vitro. But up to now, However, the mechanisms of these signaling molecular were regulated are not very clear. This study focus on the mechanism of slimb interact with Shh, wnt signal transduction in normal tooth development.METHODS AND MATRIALS This study employed cell culture, gene transfection, RT-PCR, flow cytometry,RNAi, immunohistochemistry, immunofluorescence, systematically detected slimb gene expression,β-TrCP expression in tooth germs. Acquire the mouse head of embryo day 18, postnatal day 0,3,6 respectively, 4% paraformaldehyde fixation for 48 hours, Paraffin embedding, examined using LsAB (labeled streptavidin-biotin) method, then observed theβ-TrCP protein expression pattern, and the images were analyzed. And study spectrum of gene expression after slimb-RNAi. The RNAi effects were detected by RT-PCR and Western blot, respectively. RNA extraction and semi-quantitative real-time PCR (qRT-PCR); Plasmids construction for promoter assays.RESMLTS Our data demonstrated thatβ-TrCP expressed in oral epithelium, tooth bud, mesenchymal cell cytoplasm of ameloblast and odontoblast of different stage of tooth development. And these findings provide the evidence of antagonist regulatory pathways for Shh in teeth development. Andβ-TrCP were characteristically expressed in the cytoplasm of hair stem of hair follicle, hair cuticle, cuticle of root sheath, Huxley’s layer of internal root sheath cell, external root sheath and mesenchymal tissue, respectively, whereasβ-TrCP were negatively expressed in connective tissue sheath, Henley’s layer of internal root sheath.The study of RNAi for slimb/β-TrCP demonstrated that once the slimb/β-TrCP were silenced , GLI1,GLI3,β-catenin were down-regulated in tooth germ cells, and then promote the proliferationof odontogenic cell such as dental epithelium cells and dental mesenchymal cells. And there were many amounts of Undifferentiated cells appeared. The phenomenon is similar in epithelial cells and mesenchymal cells. The study of RNAi for slimb/β-TrCP in vivo demonstrated that in early stage of mouse development, siRNA of slimb/β-TrCP can lead the mouse to death. But, in later stage of mouse development, siRNA of slimb/β-TrCP affect tooth development slightly. If siRNA of slimb/β-TrCP were injected belly cavity before the bell stage of tooth development, the tooth development would arrest at bell stage, If it were injected after bell stage, tooth development were affected slightly. CONCLUSION Our data demonstrated that slimb regulate tooth development via key signaling pathway such as sonic hedgehog signaling pathway and wnt pathway. The mechanisms of regulation were controlled by cell cycle alteration and apoptosis of dental cells after B-TrCP RNAi. The slimb gene RNAi induce enamel enlargement and Shh RNAi result in apoptosis of immature tooth epithelium cell, as same as dental follicle cells. In conclusion, although the mechanisms of tooth development are sophisticated but slimb gene regulate different other gene expression may be the important pathway.Based on these findings,β-TrCP expressed during from early stage to later stage of murine tooth development, we conclude that the slimb/βTrCP have important role in tooth development. It regulate tooth development via sonic hedgehog, wnt signaling pathway. Andβ-TrCP expressed characteristically of developmental hair follicle tissue. It’s a implication ofβ-TrCP regulate developmental hair follicle keeping normal development via mediating different signal transduction pathway.更多还原