Effects Of Amelogenin On Differentiation Of Odontoblasts MDPC-23 Cells

The differentiation of dentin cells is the crucial step of dental pulp damage repair and also is the important stage of tooth germ development. The differentiation process is regulated by many different factors. It is the hot point in searching the induced factors of dentin cells differentiation and clarify the detailed mechanisms, and is also the key point to investigate the dental pulp damage repair mechanism and discover the potential therapeutic targets. So far, it is already known that there are a great amount of factors involved in the regulation of dentin cells differentiation ,including the extracellular matrix and receptors, growth factors, homeobox genes, oncogenes, transcriptional factors, retinoic acid and receptors, cell adhesion molecules ,and so. One of the most interested research topics was the growth factor and their receptors in cell differentiation regulation and already maked much progress.Amelogenin (AMG) is one group of homologic low MW (20-30 kD) hydrophobin secreted by adamantoblast. AMG enriches proline, glutamine and histidine, which constitutes about 90% of emdogain. AMG expresses in almost all of the tooth development stages. The current accumulated research data mainly focused on the expression pattern in dental germ, the effects in enamel development and the regeneration of periodontium. Tompkins reported that the purified rat AMG protein promoted the maturation of cultured rat dentin cells in 2005, which suggested the differentiation induction ability of AMG in dentin cells.Therefore, the objective of this study was to observe the differentiation induction ability of mouse AMG to dentin cells MDPC-23. Moreover,study effect of AMG on proliferation and differentiation of detin cells and effect on phosphorylated protein of MAPKs pathways .Furthermore,inhibitors are used to block specific pathways of MAPKs to observe their impacts on AMG-induced detin cells differentiation.Also,it is supposed to provide theoretical evidence for further molecular mechanism study of AMG-induced dentin cells differentiation during teeth injury and self-restoration Firstly, the mouse gene AMG was cloned from MDPC-23 cell, which the cDNA full length is 591 bp and Genbank number is NM 009666. Meanwhile, we design the shRNA knockdown sequence of mouse AMG.. Then the high titer adenovirus was acquired based on the AMG overexpression and the shRNA vectors (Ad-AMG and Ad-shAMG), plus the control vector Ad-EGFP.Secondly, after adenovirus was infected the target MDPC-23 cells and test the protein expression changes. The results showed that AMG overexpression induced the MDPC-23 cells with longer protrusion and cell sizes shrinkage, which implicating the energetic cell division. However, shRNA knockdown of AMG didn't induce obvious cell morphology alteration. Based on MTT assay and flow cytometry analysis, AMG inhibited the MDPC-23 cells growth was found. But shRNA knockdown of AMG didn't affect at all. FCM showed that AMG overexpression increased the cell cycle G1 phase and delayed the S phase. Nevertheless, AMG knockdown caused the opposite alteration of cell cycle. The dentin cells differentiation status was detected by Alkaline phosphatase (ALP) activity and mineralization. Herein, AMG overexpression promoted the ALP activity and mineralization ability. the increase of differentiation marker DMP-1 and DSPP following the AMG induction were detected by RT-PCR. Generally, AMG shRNA demonstrated the opposite effect to ALP activity and cell differentiation compared to AMG overexpression. Western blot results showed that AMG can promote the differentiation of odontoblast-like MDPC-23 cells via ERK1/2?p38 and MAPK pathways.In order to elucidate the differentiation mechanism of dentin cells induced by AMG, the DNA microarray was used to compare the expression profile of MDPC-23 cells with or without AMG induction. The cy5/cy3 ratio as candidates screen gate was exceed 2 stand for up-regulation and less than 0.5 stand for down-regulation. Finally, 1140 up-regulation and 487 down-regulation genes were obtained. Nextly, the picked-up candidates were validated by quantitative PCR. To be summarized, our results suggested AMG can promote the differentiation of dentin cells MDPC-23. However, the detailed mechanism is needed to be clarified in the further.