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Proteomic Study on the Mesenchymal Cells during Early Mouse Tooth Development

Author: SuLiPing
Tutor: XiaoMingZhen;WangYing
School: Fourth Military Medical University
Course: Oral medicine
Keywords: Tooth development Mesenchymal cells Proteomics Laser capture microdissection ( LCM )
CLC: R78
Type: PhD thesis
Year: 2008
Downloads: 192
Quote: 0
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Abstract


Early tooth development is an important stage of tooth positioning and stereotypes. Numerous studies show that the starting period of tooth development to determine the position of the tooth germ formation; cap stage determines the type of tooth development, a variety of biological factors in epithelial - charge plays an important role in regulating quality. Proteomics is the emerging discipline of a major study of the composition of the proteome composition and biological function, which can at the protein level comprehensive analysis of the impact of the regulatory mechanism of tooth development. Therefore, this experiment by laser capture microdissection to obtain the mesenchymal cells in the early mouse tooth germ development (start of E11.5 and the cap stage E15.5 days) mandibular molars, the use of two-dimensional electrophoresis, multi-stage mass spectrometry and the proteome research programs database query, analyze the mouse molars during the early mesenchymal cells contained protein changes in the number, type, and to further explore the regulation of factors that affect tooth development, in order to seek and find the key factor, The tooth development research provides a new way of thinking. Objective: 1. Proficiency in the developing mouse tooth germ initiation stage, bud stage, the cap stage, bell stage frozen section preparation method; master laser capture microdissection and capture a sufficient amount of cell samples ( E11.5 and E15.5 days mandibular molar mesenchymal cells); (3) to obtain two sets of cell protein Proteome identified protein spots, explore the two periods affect the molecular mechanisms of tooth development. Methods: 1. Accurate development days and C57 fetal rat preparation of mouse mandibular molars in frozen sections; 2. PixCell IIe laser capture microdissection system for frozen section on the development of the mesenchymal cells separation, collect a sample of cells to the requirement; 3 first isoelectric focusing (IEF), the second two-dimensional electrophoresis diagram of the two groups of cell samples to sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained with silver nitrate, image analysis software protein spots using high performance liquid chromatography - tandem mass spectrometry for protein spots of a secondary spectrum, as well as protein amino acid sequence, proteome database to retrieve and combine different protein spots molecular information comprehensive analysis of differential proteins were identified. Results: 1. At E11.5, 12.5,13.5,14.5,15.5,16.5,17.5 day mouse mandibular molars in frozen sections; obtained by laser capture requirements of the two groups (E11.5 tooth germ start of E15.5 tooth germ cap stage) mandibular molar mesenchymal cells; 3 two groups of cells in two-dimensional electrophoresis Figure 20 protein spots were identified of which 15 points. Mass spectrometry results show that: E11.5 cells added the protein annexin A2, actin protein beta, and pyruvate kinase M; E11.5 upregulated protein subunit of ATP synthase d, ATP synthase alpha subunit, transketolase, beta enolase; E15.5 cells new protein eukaryotic translation elongation factor 2, lactate dehydrogenase B, M-Septin; E15.5 cells increase protein eukaryotic translation elongation factor 1γ Nucleus Kernel ribonucleoprotein H2, the Nucleus Kernel sugar nucleoprotein X, lactate dehydrogenase A, a serine / threonine protein phosphatase 1γ. Conclusions: 1. Vaginal plug observation and fetal rat height measurement method to improve the utilization of gestational age to determine the accuracy and mice; After consecutive frozen sections on the various stages of the development of fetal rat head clear the specific location of the mandibular molars, greatly improving the efficiency of the tooth germ frozen sections; 3. successful target cells by laser capture microdissection; through the identification and analysis of protein spots, protein obtained for cells glucose metabolism and energy metabolism protease involved in the synthesis of protein translation, and signaling molecules involved in some of the signal transduction pathway during tooth development starter and cap-like mesenchymal cells in the presence of specific expression of these proteins in mouse mandibular molars or expression, which provides new clues for further study the mechanism of regulation of the early development of the tooth germ.

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