Initial Research Of The Caveolae/Caveolin In Tooth Development Of Mouse

Since 90's in 20 centuries, along with the fast development of the foundation courses, such as cell biology, molecular biology, immunology and genetics...etc.and stem cells and the tissue engineering techniques in the modern medical science foundation study and clinic of application, the reborn medical science is already initial to display the good development foreground, making tooth regeneration to make possible gradually, causing more and more scholar's concern to this field. At present, the research of tooth regeneration is focused on which the molecular signal regulate for odontogenesis. It is one of the most important physiology functions of caveolae/caveolin-1 that the cell signal transduction. The caveolae is the terrace for the cell signal transduction and making cross talk of each signal thoroughfare be possible. The caveolin-1 is placed in that terrace in the center position of the each signal thoroughfare.The researches of developmental biology indicated that the Cranial Neural Crest Cells (CNCCs) contributed significantly to the formation of craniofacial structures during embryonic development. During this process, CNCCs underwent two key cascade differentiation, CNCC to the first branchial arch ectomesenchyme to oral and maxillofacial ectomesenchyme. This study was aimed to isolate and culture the ectoderm mesenchyme cells from the first branchial arch and the dental papilla mesenchyme cells of the E9.5 and E16.5 mice embyos, and to confirm the molecular and cellular characterization of ectomesenchymal cells and dental papilla mesenchyme cells during multilineage differentiation in vitro. At the same time, Adopt the molecular biology technique to provide the basis of theoretics for regulation mechanism of tooth development furthely. There were four parts in this thesis:1. To isolate and culture the ectoderm mesenchyme cells and the dental papilla mesenchyme cells of mice embyos in vitro. Firstly, the first branchial arch primordia from E9.5 embryos and the mandible molar papilla from E16.5 embryos were dissected under microscope. Explanted culture and improved enzyme digestion were all used to primary culture. The growth curve, population doubling time, cells proliferation and morphology were studied, immunocytochemistry assays were used to identify the source and the state of the ectoderm mesenchyme cells and the dental papilla mesenchyme cells. Secondly, monolayer cultures of mesenchyme cells were passaged 5 times and then transferred to adipogenic,endothelial and osteogenic media by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by reverse transcription/Polymer-rase chain reaction technique.2. Observe the expression of caveolae/caveolin-1 in tooth development of mouse. On the foundation of above-mentioned trial, observe the change of caveolae of ectoderm mesenchyme cells and the dental papilla mesenchyme cells in different development period by transmission electron microscope and flow cytometry. Using immunohistochemistry and RT-PCR technique to study the expressidon of caveolin-1 mRNA in tooth development of mouse with different mesenchymal cells. This help us to understand the interaction relation between the caveolin-1 and the differentiation of ectoderm mesenchyme cells.3. This study is to construct recombinant adenoviral vector carrying the mouse caveolin-1 gene using the recombinant adenoviral vector system AdEasy. The cDNA fragment of caveolin-1 was derived from pTRE2-caveolin-l by restriction enzyme and subcloned into shuttle plasmid pAdtrack-CMV. The resultant plasmid pAdtrack-CMV-caveolin-1, after linearized by digesting with restriction endonuclease Pme I, was transformed into E. coli. BJ5183 had been transformed by adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd-caveolin-1 was screened by alternation of kanamycin and then identified by restriction enzyme. The linearized adenovirus plasmid pAd-caveolin-1 was packaged in 293 cells, then, Ad-caveolin-1 was harvested. The caveolin-1 gene recombinant adenovirus can be using to study of gene function and gene-modified for stem cell in tissue engineering, imitate the time-space particularity expression of caveolin-1 on the dental development in vivo.4. This study is to evaluate the feasibility of Ad-caveolin-1 gene transfection into ectoderm mesenchyme cells and the dental papilla mesenchyme cells. The overexpression of caveolin-1 genes promote the change of expression of cyclinD1 were detected by flow cytometry. The expression of odontogenesis relatively cyclin D1 genes of these cells were detected with RT-PCR technique. To assurance Whether caveolin-1 participated the regulation of cell propagation by cyclin D1.The result of study enunciation that the first branchial arch in E9.5 were very evident and easy to dissected. Improved enzyme digestion of primary culture had less epithelial-like cells, the cells were grown to confluency for 2-3 days. The growth curve, population doubling time and morphology of two methods after passage were similar. Anti-CD57 were all positive. Observe the change of caveolae of ectoderm mesenchyme cells and the dental papilla mesenchyme cells in different development period by transmission electron microscope and flow cytometry. Using immunohistochemistry and RT-PCR technique to study the expressidon of caveolin-1 mRNA in tooth development of mouse with different mesenchymal cells.This help us to understand the interaction relation between the caveolin-1 and the differentiation of ectoderm mesenchyme cells. The overexpression of caveolin-1 genes promote the change of expression of cyclinD1 were detected by flow cytometry. The expression of odontogenesis relatively cyclinD1 genes of these cells were detected with RT-PCR technique. Proveed the caveolin-1 participated the regulation of cell propagation by cyclinD1.In conclusion, culture model of ectoderm mesenchyme cells and the dental papilla mesenchyme cells was successfully established in vitro. Sufficient and high purity ectomesenchymal cells could be harvested in short time by improved enzyme digestion primary culture, ectoderm mesenchyme cells and the dental papilla mesenchyme cells had high activity of proliferation and self-renewal and were capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research and the orofacial development. By construction recombinant adenoviral vector carrying the mouse caveolin-1 gene using the recombinant adenoviral vector system AdEasy. Proveed the caveolin-1 participated the regulation of cell propagation by cyclinD1with transmission electron microscope , flow cytometry , immunohistochemistry andRT-PCR technique.