Study Of Cbfal On The Tooth Development And Mineralization

Tooth development and mineralization is constituted by the interactions between epithelial-mesenchymal cells and tissues. At the molecular level, these interactions involve complex signaling networks composed of growth factors, their receptors, signal molecules, transcription factors and extracellular matrix proteins. Transcription factors can control the transcription of their target genes when they bind to the DNA-binding sites on their promoters. They can transfer the messages of growth factors and so on to those effective molecules. Core binding factor al (cbfal) is such a kind of transcription factor, which can control the differentiation of osteoblasts peculiarly. They can induce the pro-osteoblasts or non-osteoblasts to synthesize and secrete most mineralize related proteins such as ALP, OC, OPN, BSP and so on, hi order to differentiate to osteoblasts finally. Some foreign researchers found recently that cbfal is also expressed during tooth development, and not only whole bones development but also the tooth development are abnormal in cbfal knock-out mice. They found there are two cbfal binding sites on the promoter of ameloblastin gene which is only expressed in tooth germs. We discuss the effect of cbfal on tooth development and mineraliztion by applicationDepartment of Operative Dentistry and Endodontics FMMUwith cells culture in vitro, PCR method, gene reconstruction, gene transfection, immunohistochemistry, antisense technology et al. We also discuss the effect of cbfal on mineralize related genes transcription and the modulation of cbfal controlled by a few growth factors in dental papilla cells in vitro. We hope the molecular mechanism of tooth development and mineralization will be clear on transcriptional level, so as to give more groundwork for next research.First of all, we cloned the complete encoded sequence of mouse cbfal gene from mouse tooth germ successfully and proved that cbfal gene is expressed in tooth germs. According to the structure and open reading frame of cbfal gene, we selected a 204bp segment comprised of part of runt domain and nucleus location signal and cloned it into expression vector pRSET A. The recombinant plasmid was transformed into E.coli BL21 and induced by IPTG to express fusion proteins. The recombinant proteins were purified by metal-chelate affinity chromatography on Ni-NTA resin. SDS-PAGE showed that a new protein band about 14KD was gained and purified. Complete Freund's adjuvant was used when immunized the rabbit with recombinant Cbfal. The specific IgG antibody was feuther purified with sulfuric acetate. Total proteins isolated from mouse tooth germs, liver and skull tissues of mouse different developing stages were used to proceed Western blotting study. Meanwhile, immunohistochemical techniques were also applied to examine the antibody. All experiment above showed the antibody we prepared was correct and specific to cbfal proteins and the effective deepness was 1:400 in immunohistochemistry and 1:1000 in Western blot.Secondly, immunohistochemical techniques were applied again to examine the cbfal specificities on dental papilla cells cultured in vitro and tooth germs hi different develop stages. It was found that cbfal was detected both in dental epithelium, mesenchyme and in dental sac during cap stage germs. It wasDepartment of Operative Dentistry and Endodontics FMMUupregulated in ameloblasts and downregulated in dental mesenchyme during bell stage germs, but was found in dental papilla cells under odontoblasts. During secretion stage, there was no cbfal proteins in odontoblasts and few cbfal positive signals in dental papilla. But it was strongly expressed in ameloblasts and dental alveolar cells at the same tune. It inferred that cbfal might play a key role during tooth development especially during the differentiation of odontoblasts and ameloblasts. We also found that neither cbfal mRNA nor cbfal proteins is expressed hi...