【作者】 程敏；

【导师】 孙宏晨；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2008， 博士

【摘要】 目前国内外对牙发育调控机制的研究已由细胞水平深入到了分子水平。一些生物分子以不同的时空表达模式在分子水平对牙发育的过程进行调控,主要包括信号分子、信号分子受体和转录因子等。这些分子形成了调节牙发育的信号网络系统。学者们对该信号网络系统进行了较多的研究工作,然而迄今为止有关此调控的分子机制尚不十分清楚。对牙发育机制方面的研究,常选用动物牙胚作为研究器官发育、形态发生和细胞分化的经典模型。以往的研究多选用大鼠、小鼠和犬的牙胚作为实验模型,近年来逐渐有报道采用人牙胚作为研究对象,但报道较少。本实验分别收集了人牙胚、出生后乳鼠及犬乳恒牙替换期组织学标本,常规切片制片,对牙胚发生、发育过程作系统观察,采用免疫组织化学方法,研究TGF-β1、BMP-2、bFGF、E-Cadherin四种生物分子在牙胚发育过程中的时空表达模式及可能的调控机制。本研究结果表明;TGF-β1、BMP-2、bFGF、E-Cadherin在牙胚发育的不同时期均有不同程度的时空表达,且相互之间存在着一定的时间和空间上的一致性和种属之间的一致性。它们在牙胚发育的早期及晚期均有不同程度的表达,说明在牙冠、牙根及牙周组织的发育中均参与了牙胚组织细胞增殖、分化及硬组织的矿化,参与了牙胚发育的分子调控。同时,本文还对口腔硬组织切片和组织化学染色及其应用进行了研究。针对牙和骨骼硬脆致密,难切易碎,牙龈、牙周膜和牙髓易受损伤分离及常规切片所出现的问题,对制片技术和染色方法进行了改良。改良方法制作的牙齿、骨骼、牙及牙周组织切片和常规HE染色,镜检各种组织和细胞形态结构清晰,切片质量和染色效果优良。本研究弥补了国内目前尚无口腔硬组织切片组织化学染色的空白。目前,改良的组织化学染色法,已逐渐用于牙发育、骨折愈合、骨缺损修复、颞下颌关节损伤、腭裂、正畸、种植、口腔材料和药物等实验研究,并取得了满意结果。更多还原

【Abstract】 1.The expression and the significance of TGF-β1,BMP-2,bFGF and E-Cadherin in tooth developmentFrom embryonic oral epithelium genesis,development,to deciduous teeth eruption and in the entire process of permanent tooth replaced deciduous tooth,there is not only concluded the interaction between the cells and tissue, but also many material molecule took part in regulation.The morphology and molecular regulation mechanism on every stages of tooth development is very complex,some of these are not entirely clear at present.On the molecular regulation of tooth development immunohistochemical study has been widely reported,may be affected by the sources restrictions,the research of animals tooth development were more for example the rats teeth development,but the reports of human tooth development in immunohistochemical were less.TGF-β1,BMP-2,b-FGF,E-Ca had important roles on the regulation on different stages of tooth development.So we study the the expression of them in the human fetal dental germ,after birthed of suckling mice and dogs during the process of permanent tooth replaced deciduous tooth,to identify their molecular regulation role and significance on different stages of tooth development,and provide experimental basis for clinical application for an in-depth study of the tooth development,mineralization process mechanism, dental tissue engineering,dental structure,morphology,development,eruption and abnormal shedding(1)The morphological and molecular regulation of human fetus teeth development in every stage41 cases of human fetal tooth germ tissues which conceptus age of 8-36 weeks,were conventional paraffin sectiond,HE stained,special staining and immunohistochemical staining,under light microscope observed the histology and embryology of the morphological characteristics of human fetal tooth germ from epithelial occurred,the bud stage,cap stage,bell stage to sclerous tissue formation,and observed the expression of TGF-β1,BMP-2,b-FGF, E-Ca during the period of tooth development by immunohistochemistry The results were that:TGF-β1 in every stages of tooth development all have specific time and space distribution.In early tooth development,the positive expression of TGF-β1 in inner enamel epithelium,outer enamel epithelium,and stratum intermedium so that TGF-β1 participate in the molecular regulation of early teeth development.In the later tooth development,the expression of TGF-β1 in ameloblasts and odontoblasts cells strongly demonstrated that TGF-β1 was related with proliferation and differentiation of ameloblasts and odontoblasts cell.In the bell advanced stage,TGF-β1 was positive expression in the dental follicle,which can surmise TGF-β1 took part in the tooth root formation and eruption.Bone morphogenetic proteins(BMPs),except BMP1,belongs to TGFβsuperfamily members,the BMP signal transduction was similar with the TGF βfamily members of signal transduction.In early human tooth development BMP-2 had strongly positive expression in tooth mesenchymal cells,BMP-2 was strong expression in odontoblast and preodontoblast in the late bell stage, the same as mature ameloblast,which suggests BMP-2 was concerned with proliferation and differentiation of odontoblast and ameloblast.The positive expression of BMP-2 in the dental follicle,illustrated that BMP-2 may be participated in the molecular regulation of root development and tooth eruption.The results of this study show that b-FGF mainly distributed in human dental germ bud stage enamel organ epithelial cells and bell stage mature ameloblast,odontoblast,cap stage enamel organ inner and outer ameloblast and the dental papilla cells also showed strongly positive expression,illustrated in the human tooth growth and development,the tooth bud stage and cap stage was the major period of b-FGF reacted.At the same time,this study also found that b-FGF expressed in the human embryo dental papilla cells,in the bell intermediate and stage has the strong to weak trend.E-Cadherin was expressed in human dental germ from bud stage,cap stage to the early bell stage,mid bell stage,bell advanced stage,with the cell differentiation and dental germ morphogenesis,the positive expression gradually increased,illustrated E-Cadherin took part in the development of dental germ,the dental morphogenesis,cell differentiation,in particular it plays an important role on tooth development.The strongly positive expression of E-Cadherin in ameloblast,odontoblast of mid-bell stage dental germ,suggested that E-cadherin was concerned with enamel,predentin early development.In the bell advanced stage,cuspis of tooth emerged mero-mineralizated,mineralizated of enamel,dentin was negative,but ameloblast cells,odontoblasts which close with the hard tissue formation was strongly positive staining,suggesting that E-Cadherin was closely related with the development and mineralization of enamel and dentin.(2)The expression and significance of TGF-β1,BMP-2,b-FGF,E-Ca in tooth development of morphologyPostnatal 1-22 days neonatal rat,which the shape of teeth were bell advanced stage,sclerous tissue formation,eruption stage.TGF-β1,BMP-2, bFGF,E-Cadherin in postnatal neonatal rat dental germ development,TGF-β1 mainly expressed in odontoblasts,with the growth of tooth development gradual enhanced;BMP-2 and E-Cadherin mainly expressed in the pre-dentin, BMP-2 in the neonatal rat tooth development,the expression gradually weakened in the later stage in tissue;The expression of b-FGF in the neonatal rat tooth germ mainly in the inner enamel epithelium,outer enamel epithelium, dental follicle,stellate reticulum which had the same trend of human embryo.(3)Deciduous teeth eruption,amotic of dogs and the process of permanent tooth replaced deciduous tooth,with the expression and significance of TGF-β1,BMP-2,b-FGF,E-CadherinThe deciduous teeth of six weeks old dogs have erupted;at 3-4 months there had the root absorption,bone remodeling,the alveolar bone absorpted and bone deposition;five months old deciduous teeth lost and permanent teeth eruption.TGF-β1,BMP-2,b-FGF,E-Cadherin expressed in all predentine of dog’s permanent germ,b-FGF expressed in ameloblastoma,odontoblast is much more obviously,but TGF-β1 or E-Cadherin in these two cells without expression,disaccord with human tooth development.TGF-β1,BMP-2,bFGF, E-Cadherin in stratum intermedium and dental follicle were positive expression.BMP-2 expressed in the periodontal ligament fibers,osteoblast, bone cells and bone marrow of dogs’ deciduous teeth periodontal tissue strongly,illustrated that BMP-2 participated in the periodontal tissue and bone development.The positive expression of b-FGF in the ameloblast cells and the epithelial root sheath strongly in dogs’ permanent germ and in periodontal tissues,shows that b-FGF participated in the development ofperiodontitis.The innovation of this research:①This study observed the entire process of morphology about dental germ developing,deciduous teeth exuviation,the eruption of permanent teeth and molecular regulation.②May be because of specimens sources was restricted,the reported on tooth development more about rats and other animals research,on the human tooth occurred and the every development stag,the morphology and molecular regulation was less reported.This experiment collected different stages specimen of tooth tissue, observated human dental germ development of the organization during the morphology and molecular regulation.③The morphology and molecular regulation on deciduous teeth resorpted exfoliated and permanent tooth replaced deciduous tooth was not clear,and the mechanism of deciduous teeth loss and eruption was in the theory stage,the study of permanent germ development and molecular regulation has not yet been reported.2.Exploratory development of oral sclerous tissue slice and specific stainIn order to enhancement the quality and the effect of stain in tooth,bone and tooth with periodontium slice which usually used in oral histopathology and other stomatology empirical study.There are so many problems of practice slice and staining,for example the tooth and bone are tight firm and brittle,and difficult to cut and fragile,gingival and periodontal ligament,pulp are traumatized easily.Our study has a satisfactory result in slice technique and specific stain method and application.(1)The investigate in elevating the quality of oral sclerous tissues slice ①First,fresh specimen fixation need sufficient as early as possible.The commen soak fixation can’t soak into tooth tissue and bone tissue quickly, fixation badness even autolysis and putrefaction.The improving method used in the specimen of human and animal which has fixative in short time by decompression fixation,experimental animal blood vessel perfusion fixation, is grounding and premise in elevating the quality of slice and the result of staining.②The fomer method of decalcification is one-time cut block for biopsy tissue,but this may injured the tissue,and it is difficult to get small parts which need to observe,not cut or incomplete cut.So,a new special method of tiered decalcification was established,and repairs cut,selected the section,markers, directional embedded.③This techniquce created the method for decalcification which used both formic acid aluminum chloride and EDTA which have’t reported in the literature yet.In the past,the use of nitrate to decalcification damaged tissue and cell morphous and material in nature,with bad stain or even no stain.A new modified method changed strong acid to formic acid and used EDTA decalcificated well but slowly.This decalcificate solution is the key of this special technology which Improve the quality and stained effects.④The general tissue universally used alcohol particularly high concentration of alcohol to dehydrate,xylene to transparence,and tissue was easily contracting,hard and brittle,inaptitude in oral hard tissue.So change alcohol to n-butyl to dehydrate,used chloroform for transparent,and avoid shrinkage hard and brittle which will help for sections.⑤The modified method used liquid wax from low melting temperature to high,dipped in liquid wax for full time,slightly higher than the melting point of paraffin’s the critical temperature,used the rosin of paraffin with glue embedded,which as hardness as teeth and bone,avoid tissue fragmentated, sliced fine.⑥Extended slice,sticks and broil slice:cutting from root to dental crown,uniform and slowly,3-4μm slices were prepared,followed by 40% alcohol and showed the slice in warm water,stick on the clean glass slide which has glue on the surface,70℃for 6h-baked overnight.⑦Result of the drum dyeing and the test under microscope:oral cavity sclerous tissues section what were made with the improvement method above-mentioned were colored with HE dyeing convention,under microscope the morphosis of various kinds of tissues and cells were distinct,slices were complete,there have neither obviously chip nor other factitiousness change from the process of the section making,and the dyeing of the sections are bright-colored,anti-tartaric acid ACP histochemical stain showed that the odontoclast,the osteoclast and the uninuclear pro-osteoclast were red,the nucleolus were blue while the background were green to undergo afterstain with hematine and bright green.(2)Specific stain method of oral sclerous tissues section and the exploratory developmentSome of the structure of tooth and periodontal tissue are multiplicity,so section need specific stain besides the HE dyeing.Because the HE dyeing make the nucleolus blue and the cytoplasm red,different tissues to recognize each other main from shape,enamel matrix and dentinal matrix of the intermediate and advanced stage tooth germ were special tissues without cell structure,but the HE dyeing were simple red what could not distinguish these tissues.So we need the specific stain. A special stain of oral cavity sclerous tissues section have no report until now,we improved the Gomori special stain and used for the special stain of oral sclerous tissues section.The improved special stain could distinguish the enamel matrix and dentinal matrix of the intermediate and advanced stage tooth germ,bone and cartilage,the sclerous tissues and the soft tissues of tooth and periodontium with bright showy red and green,the nucleolus were showed between blue and amethyst,the morphosis of various kinds of tissues and cells were distinct.This method especially suitable for the learner and a nonprofessional staff of pathology.To recognize and distinguish the tissues from color was easy to observe and research,and image analysis quantitative study could be carry out.The dyeing result was not easy to fade and it was good for making teaching section and doing retrospective study.The effect of photographing photochrome and media mix all were excellent.The improved specific stain method was used in the research about development of tooth,union of fracture,restore of bone defect,damage of TMJ,fissura palatine,periodontitis,oral materials and medicine,and so on. The method was gained satisfactory consequence.更多还原