| Tooth development and mineralization is regulated by the sequential and reciprocal interactions between the epithelial and mesenchymal tissues ,Which is composed of complex signaling network, including growth factors, their receptors, transmembrane signal transduction, transcription factors and intracellular signal pathway. Theses pathway induce some special gene expression and affect some special biological behavior of these cells. As a special biomineralized organ, tooth development is an important model of these procedure. The extracellular matrix proteins secreted by ameloblast and odontoblast are the key protein for the development of enamel and dentin. Amelogenin is the most abundant extracellular matrix protein in enamel developing. Amelogenin-deficiency can cause amelogenesis imperfecta(AI). What is the reason of the cell specific gene expression? The most important thing we care of is transcriptional regulation. So to find the key trancription factors is very important for us to learn the transcription information. Cbfal is a strong regulator for osteo-specific gene expression, such as ALP, OC, OPN, BSP. The Cbfal-deficient mice also showed abnormal dental development. According to the expression characteristic of Cbfal in tooth development. We soppose that Cbfal can regulate the trancription activity of Amelogenin. To prove the suppose, we did the experiment as followed.Fitst, the 8.5 kbp secquence of 5'flanking of Amelogenin gene was anylized,and some potential Cbfal binding sites were found. According to the most common core binging secquence-PuACCPuCA. 3 binding sites at -5224, -1641 and +92 were found. To analyse all the transcription region, we amplified the overlapped secquence3760bp and 3035bp by PCR from the genomic DNA of mouse C57BL/6J. By restriction endonucleases enzyme digestion and ligation, 5 transcriptional regulation sequences different in length of the 5'flanking of Amelogenin gene including the basal promoter were cloned and ligated with luciferase gene in PGL3 -Basic vector.These report genes were transient transfected into CHO, Hela and UMR-106 cells and luciferase assay was performed to analyze the transcription activation of these promoters. The activity of luciferase was very strong in Hela cells. On the contrary, the CHO and UMR-106 cells showed weak fluorescence. Hela cell can be used as a good model to study the transcriptional regulation of Amelogenin promoter. According to the different activity of different length, it is suggested that there were some potential siliencer located between -1693 and -975, and some potential activator in the region between -532 and -285. The longest promoter report gene was co-transfected into Hela, CHO, and UMR-106 cells with Cbfal to compare the different roles of Cbfal in Hela, CHO, and UMR-106 cells. Cbfal served as a strong transcriptional activator on Amelogenin promoter in Hela cells. In CHO and UMR-106 cells, Cbfal showed weak activation. It proved that this activation is epithelium-specific. To find the binding site of Cbfal in the promoter region, the activitation of Cbfal was systematically tested in Hela cell by co-transfection of Cbfal with 6 different Amelogenin promoter report genes. In this way, we found that activation of Cbfal on single site didn't upgulate in large-scale . It seemed that the up-regulation is related to cooperation of muti-sites . To confirm the conclusion of bio-information analysis, three report vector deleted or mutated on -5224, -1641 and +92 are constructed. Co-transfected with Cbfal to Hela cells, the three sites showed different activation. -1641 and +92 are concerned with the up-regulated expression. -5224 showed weak activation.To further prove the two site -1641 and +92 were the binding sites of Cbfal. |
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