Preliminary Study On The Effect Of Cdc42 In Mouse Enamel Development

Background and objection:In the process of enamel development, the ameloblasts can not only synthesize and secrete enamel matrix, but also reabsorb and degrade them. At the same time, the cells are associated with the active calcium transport. The ameloblasts can be called the key cells during amelogenesis. As the external environments or internal conditions such as nutrition and genetic factors change, enamel hypoplasia could happen because the ameloblasts have abnormal cell differentiation. Many domestic and foreign scholars have explored the ways for culturing amelobasts, which lay the foundation for studying enamel development in vitro.Amelogenesis imperfect (AI) is due to the malfunction of genetic factors without other diseases of the whole body. People afflicted with amelogenesis imperfecta have teeth with abnormal color:yellow, brown or grey; this disorder can afflict any number of teeth of both dentitions. The teeth have a higher risk for dental cavities and are hypersensitive to temperature changes as well as rapid attrition, excessive calculus deposition, and gingival hyperplasia. The studies of AI in our country mostly focus on case reports at present, while the foreign scholars have explored in great depth the pathogenesis of AI.Amelogenin was the first factor found to cause AI. More and more genes have been found soon afterwards. Mutations in the Amelogenin (AMELX)、Enamelin (ENAM), Matrix metalloproteinase 20 (MMP20), Kallikrein 4 (KLK4)genes have been found to cause amelogenesis imperfect. The mutation number are 15,9,4 and 1 respectively. However, they are just a quarter of the whole AI family number, which means that there are more other AI disease related genes. Many explorers have studied more on it. Researchers expect that mutations in further genes are likely to be identified as causes of amelogenesis imperfecta.Recent studies have highlighted a potential regulatory role for Rho-mediated signaling in amelogenesis. The Rho family of GTPases is a family of small signaling G proteins, and is a subfamily of the Ras superfamily. According to the sequence of cognate degree and function, it can be divided into four categories including RhoA, Racl, Cdc42 and lack of GTP enzyme activity. Three members of the family have been studied in detail:RhoA, Racl and Cdc42. GTPases are molecular switches that use a simple biochemical strategy to control complex cellular processes. The cycle between two conformational states:one bound to GTP (active state), the other bound to GDP (inactive state), and they hydrolyse GTP to GDP. The members of the Rho GTPase family have been shown to regulate many aspects of intracellular actin dynamics, and play an important role in cell polarity, microtubule dynamics, membrane transport and transcription factor activity. In other words, they involve in the processes including cell polarity, cell movement, cell shape, cell-cell interaction, cell swallowing and exocytoses and so on.As a member of the Rho GTPase family, the cell division cycle protein 42 (Cdc42) acts as binary molecular switch that is turned on in the GTP-bound state and turned of in the GDP-bound state in response to a variety of stimuli. Cdc42 has a conserved role in regulating cell polarity and the actin cytoskeleton in many eukaryotic organisms. Cdc42 has been shown to have a role in yeast budding, epithelial polarity, migratory polarity and fate specification during cell division. Cdc42 can induce reconstruction of pseudopodia, and the polymerization and assembling of actin, so as to maintain and reconstruction the cytoskeleton. At the same time, Cdc42 may participate in regulating anchoring dependence cell cycle by the function of p21CIPI. It plays an important role in the process of transcription caused by extracellular factor stimulating. In addition, Cdc42 molecules can contribute to cell swallow, which often happen in some special micro environment.There are no information about Cdc42 and tooth development at present, especially the expression and function of Cdc42 in enamel development. Thus, the researcher mainly focused on the expression of Cdc42 during amelogenesis and the probable effect during this process.The paper consists of the following four partsChapter 1 The expression of Cdc42 during mouse dental germ developmentEmbryo mice of E13.5, E15.5, E17.5 and E19.5 were selected. The dental germs of each stage were obtained. The serial sections were taken throughout fixation dehydration、transparency and embedding. The serial sections were stained by HE to observe the dental germ stage according to morphological criteria. Immunofluorescence was used to judge the expression of Cdc42 during the whole tooth development including the bud stage, cap stage and bell stage, which can help analyze the relationship between Cdc42 and enamel development.Chapter 2 Culture、purification and identification of mouse ameloblasts in vitroMouse ameloblasts could be cultured by enzyme digestion, which can shorten the culture time. The amelobasts were cultured in BME-coated dishes, which was beneficial to cell stick and growth. Differential digestion was used to purify the epithelial cells. The research was to explore an appropriate way for culturing mouse ameloblasts, and lay the foundation for studying enamel development in vitro.Chapter 3 The expression of Cdc42 in mouse ameloblastsRT-PCR and Western blot were used to detect Cdc42 mRNA, Cdc42 protein in mouse ameloblasts respectively. Cdc42 protein was located in cell membranes and cytoplasm by immunofluorescence in mouse ameloblasts, which lay the foundation for studying the relationship between Cdc42 and mouse ameloblasts.Chapter 4 The effect of Cdc42 supression on the expression of enamel matrix proteins in mouse ameloblastsCdc42 inhibitor ML141 was added to culture mouse ameloblasts to achieve the aim of Cdc42 supression. Western blot was used to verified the expression of enamel matrix proteins in mouse anmeloblasts, so as to explore the probable effect of Cdc42 on enamel development.Materials and methodsThe serial sections of mouse dental germEmbryo mice of E13.5, E15.5, E17.5 and E19.5 were selected. The dental germs of each stage were obtained. The serial sections were taken after fixation in 4% paraformaldehyde solution, dehydration in concentration gradient ethanol solution, transparency in dimethylbenzene and embedding in paraffin. The sections were 5μn.HE stainingThe serial sections were deparaffinized and hydrated to water. Hematoxylin was used to stain cell nucleus. Then the sections were washed in running tap water and counterstained with eosin from 15 s to 2 min depending on the age of the eosin, and the depth of the counterstain desired. Dehydrate in 95% and absolute alcohols, two changes of 2 min each or until excess eosin is removed. After dehydration in concentration gradient ethanol solution, transparency in dimethylbenzene, the sections were mounted in permount. Then check under microscope.Tissue immunofluorescence techniqueThe serial sections were deparaffinized and hydrated to water. The sections were placed in citrate buffer and hold temperature at 94℃ for 15 min, then cooled at room temperature. Treating sections with 10% H2O2 for 10 min to quench of endofenous peroxides, then remove the sections from rac、dry around tissue sections and ring tissue with a pap pen. Tissue must not dry out. Place each sections in a humidity chamber as it is prepared. Cover tissue sections with 5% BSA and incubate slides for at least 1h at room temperature. After blocking, the sections were incubated with primary antibody and then secondary antibody, Hoechst. the sections were mounted in fluorescence quenching glycerin. Then check under microscope.Mouse ameloblasts culture in vitroNewborn mice’s neck executed and soak off in 75% alcohol for disinfection. The head could be divided into two parts along the center line of upper and lower jaw. The tissues were put in cooled D-Hanks solution for cleaning. First molar tooth germs from new-born C57BL/6 mice were obtained by stereoscopic microscope.0.25% pancreatic enzyme contained EDTA were added to tooth germs, then cut up the tissue and digestion for 20 min, shaking once every 5 min to promote pancreatic enzyme action. The cell suspension was centrifuged 5 min at 1000 r/min and the supernatant was abandoned. DMEM medium with 10% FBS added and suspended the cells. The cells were cultured on a coated dish at 37℃,50% CO2 saturation and saturated humidity.Mouse ameloblasts purificationEpithelial and mesenchymal cells have different tolerance to trypsin. The cultured cells were sucked out the culture medium and cleaned with asepsis D-Hanks solution. After adding 0.25%trypsin, the cells were put under the inverted microscope to observe long spindle form fiber like cell edge contraction, and epithelioid cells had no obvious change. Then added the medium to suspend the digestion, and gently washing adherent cells. Medium was then added to culture. After a week, the digestion was repeated again.ImmunocytochemistryThe cells were cultured on the slides and fixed by 4% paraformaldehyde solution. Cover cells with 5% BSA and incubate slides for at least 1h at room temperature. After blocking, the cells were incubated with primary antibody and then secondary antibody, Hoechst. the cells were mounted in fluorescence quenching glycerin. Then check under microscope.RT-PCRThe total RNA was extracted using RNA mini Kit according to the manufacture’s protocol. Reverse transcription of total RNA was performed. The genes were amplified by PCR amplification, and ran by agarose gel electrophoresis in agarose gel. Put the agarose gel into the laser imaging system for observation.Western blotThe total protein was extracted, and the protein concentrations were measured by BCA Protein Assay Kit. The sample protein concentration was adjusted to the same level. The samples were loaded onto pre-cast 10% gels followed by electrophoresis and transfer to polyyvinylidene fluoride (PVDF) membranes for 2 h. Membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies at 4℃ overnight. After rinsing, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Finally, the developer was exposed, and the results were observed by developing instruments.StatisticsData are reported as Mean±SD. Statistical analysis of statistical data using SPSS 13.0, the measurement data with a single factor analysis of variance for comparison of multiple groups of differences, with P< 0.05 for the difference was statistically significant.Results1. The expression of Cdc42 during mouse dental germ developmentImmunofluorescence method was used to detect the expression of Cdc42 in mouse dental germs including bud stage, the cap stage, bell stage and late bell stage. The enamel oragans have different degrees of expression in enamel development.2. Culture-, purification and identification of mouse ameloblasts in vitroThe growth of the primary mouse ameloblasts was good, which was mixed with a large number of fibroblast like cells. However, the situation was improved after purification. The cells showed the shape of paving stones and highly purity.3. The expression of Cdc42 in mouse ameloblastsRT-PCR and Western blot were used to detect Cdc42 mRNA, Cdc42 protein in mouse ameloblasts respectively. Cdc42 protein was located in cell membranes and cytoplasm by immunofluorescence in mouse ameloblasts, which lay the foundation for studying the relationship between Cdc42 and mouse ameloblasts.4. The effect of Cdc42 supression on the expression of enamel matrix proteins in mouse ameloblastsThe Cdc42 inhibitor ML141 was used to culture the mouse cells to inhibit the effect of Cdc42. Western blot was detected that there were different degrees of changes in mouse enamel matrix proteins.Conclusion1. Cdc42 expressed in each stages of tooth development especially in enamel organ, indicating that Cdc42 may involve in enamel development.2. The primary cultured ameloblasts were mixed with some fibroblast-like cells. However, the purified ameloblasts were cobblestone-shaped and grew well. Conclusion:Mouse ameloblasts could be cultured by enzyme digestion, and purified by differential digestion in BME-coated dishes, which Is beneficial to the related fundamental researches.3. Cdc42 expressed in mouse ameloblasts, and located in cellular membranes and cytoplasm of mouse ameloblasts, indicating that Cdc42 has close relationship with mouse ameloblasts.4. The expression of AMELX, AMBN, MMP20, KLK4 changed when Cdc42 was inhibited, indicating that Cdc42 may affect the expression of enamel matrix proteins in mouse ameloblasts.