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Amelogenin Gene Expression in Porcine Odontoblasts

¹ Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2- 1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan;
² Department of Periodontology, School of Dental Medicine, Tsurumi University, 2- 1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan; and
³ Department of Physical Therapy, School of Health Science, Niigata University of Health and Welfare, 3198 Shimami-cho, Niigata 950-3198, Japan;

View larger version (102K): [in a new window]   Figure 1. (A) Schematic drawing of ameloblast and odontoblast tissue preparations. The secretory ameloblast layer was dissected from the inner surface of the removed enamel organ epithelia (EOE), and the maturation ameloblast layer was prepared from the labial surface of the incisor. (B) The secretory ameloblast sample was prepared from the inner surface of the EOE, which contained secretory ameloblasts (SA), stratum intermedium (SI), and stellate reticulum cells (SR). After removal of all the cells covering the enamel matrix completely, the apical portion of the tooth was cut off, and the dental pulp was removed carefully. Almost all the odontoblasts remained on the surface of predentin matrix following the removal of the pulp tissue. (C) Cells remaining attached to the predentin matrix following removal of the dental pulp. Almost all the cells on predentin matrix are odontoblasts.

View larger version (83K): [in a new window]   Figure 2. RT-PCR amplification products with amelogenin (panels A, B), DSPP (panel C), and GAPDH (panel D) specific primer sets. The amplified products were separated on a 4.5% polyacrylamide gel and stained with ethidium bromide. Lane M: molecular-size standard, x 174 HaeIII-digested (New England Bio-Lab). Lane 1, oral epithelium; lane 2, muscle; lane 3, connective tissue; lane 4, dental pulp cells; lane 5, secretory ameloblasts from the incisor; lane 6, secretary ameloblasts from the molar; lane 7, maturation ameloblasts from the incisor; lane 8, odontoblasts from the incisor; and lane 9, odontoblasts from the molar.

View larger version (21K): [in a new window]   Figure 3. Quantitative PCR of amelogenin and GAPDH mRNA isolated from secretory ameloblasts, maturation ameloblasts, and odontoblasts, which permitted calculation of the relative amounts of amelogenin mRNAs from these different tissues.

View larger version (65K): [in a new window]   Figure 4. (Panel A) Analysis of RT-PCR products with amelogenin exon 2 and exon 7-specific primer sets. PCR was carried out for each sample by 25 cycles. The amplified products were analyzed by 4.5% polyacrylamide gel electrophoresis. Lane M: molecular-size standard, x 174 HaeIII digested (New England Bio-Lab). Lane 1, secretory ameloblasts in the molar; lane 2, secretory ameloblasts in the incisor; lane 3, maturation ameloblast in the incisor; lane 4, odontoblasts in the molar; and lane 5, odontoblasts in the incisor. (Panel B) Amelogenin gene expression in erupted first molar odontoblasts. Lane 1: PCR with upstream primer at exon 5 and downstream primer at exon 6; lane 2, PCR with upstream primer at exon 2 and downstream primer at exon 7; and lane 3, PCR with GAPDH-specific primers.