【作者】 白玉娣；

【导师】 吴补领； 杨富生； 轩昆；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 牙齿是由口腔上皮和神经嵴来源的外胚间充质相互作用而形成的。人类牙齿的发育,经历蕾状期、帽状期、钟状期、牙根发育和牙齿萌出期。人类的牙齿和啮齿类动物的磨牙在牙冠发育完成以后,成釉器内釉、外釉上皮细胞在颈环处增生,形成两层上皮结构,即Hertwig’s上皮根鞘(Hertwig’s epithelial root sheath,HERS),标志着牙根发育的启动。而啮齿类动物(如大鼠、小鼠)的切牙终生不发育形成典型牙根结构,且终生不断萌出。研究认为,在切牙的末端存在apical bud结构,其中有成体干细胞,后者不断缓慢分裂增生,分化为成釉细胞,形成釉质,使得切牙不断生长。那么HERS和apical bud这两种来源于成釉器的牙上皮细胞究竟有着怎样的生物学性质,它们之间又有怎样的差异而导致形成两种完全不同的发育模式呢?本课题的目的是研究这两种上皮细胞的生物学性质,寻找其间的差异,为牙根发育机制的研究提供实验依据。本课题采用组织学、免疫组织化学、细胞培养、RT-PCR、蛋白质组学和体内种植等技术方法进行了以下四部分实验研究,主要研究内容和结果如下:第一部分apical bud和HERS组织学研究实验一:采用组织学方法观察了出生后(postnatal, PN)0-10d SD大鼠切牙apical bud和磨牙HERS的组织学结构,结果发现:apical bud位于切牙胚唇侧末端,PN0d即已形成,为三层细胞构成的上皮结构,含有内、外釉上皮,及其间的星网层细胞,星网层细胞含量非常丰富,使得apical bud成为球形结构深入末端牙乳头组织中。HERS在PN 7d SD大鼠磨牙胚颈部形成,为内、外釉上皮两层细胞构成,星网层细胞消失,而呈连续的扁平状结构。实验二:采用免疫组织化学方法观察了FGF10, Notch1, BMPs在apical bud和HERS组织中的表达。结果发现:FGF10在apical bud周围牙乳头细胞中表达,而HERS内侧牙乳头细胞表达阴性;Notch1表达于apical bud中;BMPs在HERS中表达。表明虽然有着共同的发育起源,但是一些特异的调控因子表达存在差异。提示牙根发育启动存在特异性的调控机制。实验三:采用透射电镜方法,观察apical bud和HERS超微结构。结果:apical bud中部分细胞胞膜增厚,形成桥粒样细胞连接,部分细胞中间丝丰富,并呈类似黏液上皮样细胞形态。HERS两层上皮细胞整齐排列,内层为柱状或立方状细胞,细胞之间胞膜相嵌、融合,形成紧密连接。第二部分apical bud和HERS细胞生物学性质研究实验一:通过机械分离切牙胚颈部唇侧末端和磨牙胚颈部组织,酶消化法原代混合培养,10%FBS DMEM/F12培养液连续培养,差别胰酶消化法进行细胞纯化的方法,分别培养apical bud和HERS细胞。结果发现:在10%FBS DMEM/F12培养液中原代细胞生长状态良好,增殖迅速,经3次差别消化得到纯化的上皮细胞。免疫细胞化学实验证实得到的细胞为上皮细胞。实验二:通过MTT检测、流式细胞术、免疫细胞化学、茜素红染色和RT-PCR等方法对apical bud和HERS细胞一些生物学性质进行了初步研究。结果发现:apical bud细胞表达Amelogenin, Ameloblastin, Notch1,而HERS细胞表达BMP2,4。体外培养的apical bud细胞有矿化形成能力。第三部分apical bud和HERS细胞差异蛋白质组学研究实验一:通过双向凝胶电泳实验对apical bud和HERS细胞蛋白表达谱进行研究和对比。结果发现:apical bud和HERS细胞差异表达蛋白有145个,相对于apical bud细胞,HERS细胞表达的蛋白新增36点,缺失23点,有68点表达上调,有18点下调。实验二:通过肽质量指纹谱方法对实验一得到的差异蛋白进行研究,结果:鉴定出10个蛋白质,其功能涉及细胞的氧化还原反应、酶活性等方面。第四部分apical bud和HERS细胞与牙乳头细胞团体内种植实验研究利用体外培养的apical bud和HERS细胞分别与切牙和磨牙胚牙乳头细胞重组后,细胞团种植于大鼠肾被膜下。结果发现:apical bud和切牙牙乳头细胞团形成组织结构规则的釉质和牙本质组织,HERS细胞与磨牙牙乳头细胞团形成了骨样牙本质样组织。表明apical bud和牙乳头细胞间可以相互诱导,分化出成釉细胞和成牙本质细胞,形成牙体组织,HERS细胞能够诱导牙乳头细胞分化为成牙本质细胞。更多还原

【Abstract】 Tooth development is the result of the interactions between ectoderm epithelium and mesenchyme from neural crest. Human tooth development is a reciprocal and consecutive process, which includes bud, cap, bell stages, tooth root development and tooth eruption. As human tooth and rodent molar, after completion of tooth crown, the inner and outer epithelium of enamel organ proliferate at the cervical loop and form a bilayered epithelial sheath, known as Hertwig’s epithelial root sheath (HERS), which is the initiation of root development. However, the rodent incisors grow and erupt continuously the whole life. It is the adult stem cells residing on the apical bud of the incisors that leads to the continuous growth. What reasons exist in these two epithelia, the apical bud and HERS cells, resulting in the two totaly different developmental modals? The aim of this study is to explore the biological characters and differences between the two cells, in order to provide experimental evidence for the studies on the mechanism of root development.In this study, histology, immunohistochemistry, cell culture, RT-PCR, in vivo implantation and proteomics methods were used to conduct following four sections of experimental study. The major contents and results are as follows:Section 1 Study on apical bud and HERS tissuesIn experiment 1, PN0-10d SD rats’apical bud and HERS structures were systematically observed by histological method. It was found that apical bud resided on the labial tip of the mandibular incisor, which had formed at birth. Inner, outer epithelial and stellate reticulum cells comprised the apical bud and the stellate reticulum cells were pretty populous, which caused a spherical bulge between the dental papilla and follicle tissues. At PN 7d, HERS began to develop at the molar crown cervix. The inner and outer epithelia fused and developed to HERS, and the stellate reticulum cells dissappeared, leading to a continuous and flat shape.In experiment 2, the expression of some molecules, such as FGF10, Notch1 and BMPs were detected in/around the apical bud and HERS tissues by immunohistochemistry. It was found that FGF10 was possitive in the dental papilla close to the apical bud, whereas none was existed around the HERS. Notch1 were expressed in the apical bud cells. BMPs was possitive in the HERS cells. The results indicated that some certain molecules were differently expressed by the two cells, although they came from the common origin, the enamel organ.In experiment 3, the apical bud and HERS’s ultrastructures were observed by transmission electron micrograph(TEM). Some apical bud cells’membrane thickened and formed brige-like cells connection. Some cells contained plenty middle fiber and presented as mucus-like epithelia phenotype. The bilayed HERS cells lined up regularly, the inner were column and the outer were flat cells. The HERS cells connected tightly by membrane insertion and fusion. Section 2 Study on the biological characters of apical bud and HERS cellsIn experiment 1, mechanical separation of the incisor germs’labial tip and molar germs’cervical portion, primary mixed culture by enzyme digestion and differential trypsinization were operated and the two epithelial cells were cultured with 10% fetal bovine serum(FBS) DMEM/F12. It was found that primary cells grew well and proliferated rapidly. Purified epithelial cells were obtained after three times of differential trypsinization, which were confirmed by immunocytochemistry.In experiment 2, the biological characters of purified apical bud and HERS cells were investigated primarily by MTT, flow cytometry (FCM), immunocyto- chemistry, alizarin red and RT-PCR. It was found that Amelogenin, Ameloblastin and Notch1 were expressed by apical bud cells, whereas BMP2,4 were expressed by HERS cells. Apical bud cells cultured in vitro had an ability to mineralization formation.Section 3 Comparative proteomics between apical bud and HERS cellsIn experiment 1, the proteins expressed by apical bud and HERS cells were investigated by two-dimensional electrophoresis. It was found that 145 differential proteins appeared. Compared to apical bud cells, 36 new spots appeared, 23 spots disappeared, 68 spots were up-regulated and 18 were down-regulated in the HERS cells.In experiment 2, differential proteins obtained from the above were investigated by peptide mass fingerprinting method. It was found that 10 proteins were identified, whose functions involved in the aspects of redox regulation of the cell, Catalysis of the NADPH-dependent reduction of 3-deoxyglucosone, and so on.Section 4 Study on the cell pellet of the apical bud or HERS cells combined by dental papilla cells implanted in vivoIn this experiment, apical bud cells and HERS cells were recombined respectively with incisor or molar dental papilla cells and implanted into the rats’renal capsules. It was found that apical bud and incisor papilla cells pellet developed into well-formed enamel and dentin tissues, whereas the combination of HERS cells and molar papilla cells produced bone-like dentin tissues. The result demonstrated that apical bud and dental papilla cells could induce each other, resulting in ameloblats and odontoblasts differentiation, and eventually form dental structures.更多还原