Proteomic Study On The Mesenchymal Cells During Early Mouse Tooth Development

Early tooth development is an important stage determining tooth region and type. Many researches indicated that initiation stage of tooth development determined the tooth region and cap stage determined the tooth type. Signals regulating between epithelial and mesenchymal tissues have made an very important function during the two stages. Proteomics is a new science that investigates the protein composition and function of proteome.The regulating mechanisms of tooth development can be studied on the level of proteome. Therefore in the present study, we captured the mesenchymal cells of mouse lower molar germ at E11.5d (initiantion stage) and E15.5d (cap stage) by laser capture microdissection (LCM). The differentially expressed proteins of the mesenchymal cells at the two stages above-mentioned were analyzed on quantity and quality by two-dimensional gel electrophoresis, mass spectrometry, data base querying technology. The aim of our study was to further investigate the regulating factors, even to find some new signals expressed in the tooth germ and to provide a new method on the mechanisms research of tooth development.Objectives: 1. To grasp practisedly the preparative method of the frozen sections of mouse germs at different stages(initiation, bud, cap, bell stage) during tooth development; 2. To grasp practisedly the techniqu of LCM and obtain the sufficient amount of cell samples; 3. To establish the atlas of protein two-dimensional gel electrophoresis of the mesenchymal cells in the mouse lower molar germs at the stages of E11.5d and E15.5d. To identify the differently expressed proteins and investigate the molecular mechanisms of tooth development during the two stages above-mentioned.Methods: 1. C57 pregnant mice that had accurate fetal ages were obtained and the frozen sections of mouse germs at different stages during tooth development were prepared; 2. The mesenchymal cells on the frozen sections by using PixCell IIe were obtained and sufficient cells of each sample were collected; 3. The atlas of protein two-dimensional gel electrophoresis (2-DE) of samples were obtained by isoelectric focusing electrophoresis (IEF), sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and AgNO3 staining. The differently expressed proteins were analyzied by image analysis software; 4. First, second class mass-spectrograms and amino acid sequences of proteins were obtained by high performance liquid chromatogram- mass spectrum/ mass spectrum (HPLC-MS/MS). The differently expressed proteins were identifid by protein database querying and protein information analysis.Results: 1. The frozen sections of mouse lower molar germs at E11.5,12.5,13.5,14.5,15.5,16.5,17.5d during tooth development were prepared; 2. We have obtained sufficent mesenchymal cells of mouse lower molar germs at E11.5d and 15.5d; 3. We have obtained 20 differently expressed proteins on the atlas of 2-DE and 15 prteins were identified. Annexin A2,β-actin, pyruvate kinase M were expressed specificly in the sample of E11.5d; ATP synthase subunit d, ATP synthase alpha subunit, transketolase, beta-enolase were up-regulatedly expressed in the sample of E11.5d; Eukaryotic translation elongation factor 2, lactate dehydrogenase B, M-Septin(also named Septin 4)were expressed specificly in the sample of E15.5d;Eukaryotic translation elongation factor 1gamma, heterogeneous nuclear ribonucleoprotein H2, heterogeneous nuclear ribonucleoprotein X, lactate dehydrogenase A, serine/threonine-protein phosphatase 1gamma were up-regulatedly expressed in the sample of E15.5d.Conclusions: 1. The fetal age could be judged more accuratly and the use ratio of mice could be increased by the combination of observing pessulum and measuring fetal mouse body length; 2. The regoin of lower molar germ in the head had been knowed by using the method of cutting the frozen head embed of different fetal age mice one by one section. Then the efficiency of obtaining the frozen sections of tooth germs was markedly increased; 3. We have obtained the mesenchymal cells from the frozen sections successfully by using LCM;4. Using proteomics methods we have obtained some defferently expressed proteins between two samples.By identifying , the proteins mainly comprised some enzymes correlated with glycometabolism and energy metabolism in the cells, some factors related with the protein translation and some molecules correlated with some signal conduction pathways. They were specificly or up-regulatedly expressed in the mesenchymal cells of mouse lower molar germs at initiation stage and cap stage respectively, which provided a new clue for the research on the regulating mechanisms of tooth development during early stages.