Primary Detection On Differentially Expressed Genes In Odontogenic Differentiation Of Ectomesenchymal Cells Isolated From The First Branchial Arch

The latest research of developmental biology indicated, Cranial Neural Crest Cells (CNCCs) contributed significantly to the formation of craniofacial structures during embryonic development. In this process, CNCCs underwent two key cascade differentiation, CNCC — the first branchial arch ectomesenchyme (FBAEM) — oral and maxillofacialectomesenchyme(OMFEM).This study was aimed to isolate and culture the mesenchyme cells from the first branchial arch of the fetal mice, and to confirm the molecular and cellular characterization of ectomesenchymal cells during multilineage differentiation in vitro. At the same time, the double-direction suppression subtractive hybridization (SSH) cDNA libraries of odontogenic differentiation of FBAEMCs were constructed and primary detection and analysis of differerntially expressed genes were carry out. It will provide the basis of theoretics for regulation mechanism of tooth development furthely.Firstly, the first branchial arch primordia from E9.5 embryos were dissected under microscope. Explanted culture and improved enzyme digestion were all used to primary culture. The growth curve, cells doubling time, cellsproliferation and morphology were studied, immunocytochemistry assay was used to identify the source and the state of FBAEMCs. Secondly, the activity of proliferation and self-renewal of FBAEMCs were detected by nuclear labeled with BrdU and fluorescence in situ hybridization for telomerase. Monolayer cultures of ectomesenchymal cells were passaged 3 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/polymerase chain reaction technique. Thirdly, the double-direction SSH cDNA library of the 3rd passage of FBAEMCs and the dental papillae cells isolated from 1st lower molar tooth germs were built up. Analysis of total RNA qualities, cDNA Taq I digestion, adaptors ligation efficiency, subtraction efficiency and PCR products were all studied. Finally, 23 clones were randomly selected to sequence and analyzed for homology in the GeneBank. Quantitative analysis of differentially expressed genes were carried out by real-time PCR in E9.5, E12.5, E14.5 and E16.5.The nasofrontal process, the first branchial arch and the second branchial arch in E9.5 were very evident and easy to dissected. There were more epithelial-like cells in the explant primary culture, the cells were grown to confluency for 7-10 days. Improved enzyme digestion of primary culture has less epithelial-like cells, the cells were grown to confluency for 2-3 days. The growth curve, cell doubling time and morphology of two methods after passage were similar. Anti-CD57/HNK-1 and anti-Vimentin were all positive. The high activity of proliferation and self-renewal of FBAEMCs were observed by nuclear labeled with BrdU and fluorescence in situ hybridization for telomerase. After beingcultured in an adipogenesis-inducing medium, the ectomesenchymal cells responded by the accumulation of lipid vacuoles and the gene expression of LPL. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells, and formed mineralized nodules. In addition, osteogenesis was followed by collagen type I immunocytochemica staining and ALP staining. Epithegenesis was determined by vWF immunocytochemical staining. FBAEMCs qua tester, there were 184 white clones in the forward direction subtractive libraries, FBAEMCs qua driver, there were 132 white clones in the reverse direction subtractive libraries. The double-direction libraries have high quality by results of SSH analysis. After sequencing, the repeated sequences were removed, 15 fragments were analyzed for homology, the results showed 11 fragments were part of the known genes of mouse, 4 fragments were DNA sequences and likely to unknown new genes. The results of real-time PCR of seven differently expressed genes were the same as the results of SSH. Change of clone 1 -3 ralative copies was very prominent, thereby ulteriorly demonstrate it was a new gene.In conclusion, culture model of ectomesenchyme cells isolated from the first branchial arch was successfully established in vitro. Sufficient and high purity ectomesenchyme cells could be harvested in short time by improved enzyme digestion primary culture. FBAEMCs 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. Then high quality subtractive cDNA libraries of odontogenic differentiation of FBAEMCs were triumphantly constructed by SSH for the first time. The functions ofdifferentially expressed genes were primary discussed by bioinformatics. The results of SSH libraries were validated once again by real-time PCR on the histology level. It will provide the basis of theoretics for cloning the new gene.