| The differentiation of neural crest-derived ecto-mesenchymal cells into odontoblasts, the precise mechanism of dentinogenesis and tissue engineering of dentin have not been well characterized partly because of the limited availability of odontoblast cell lines, especially derived from human being. Thus it would be ideal to have sufficient amount of carefully characterized cells with same phenotype and function of human native odontoblasts. However, the use of human odontoblasts and/or ecto-mesenchymal cells from dental papillae for basic research and therapeutic purposes has been restricted partly by their limited proliferative potential that they could divide about ten times before fully exiting the cell cycle and becoming senescent and dead. Several immortalized dental papilla-derived cell lines or odontoblast-(like) cell lines, such as MDPC-23 and MO6-G3, have been established either spontaneously or via gene (including SV-40 large T antigen and HPV 18 E6/E7) transduction, but only some phenotypic characteristics of native odontoblasts were maintained. As the proteins encoded by the virus disrupt many cellular pathways, transformed cellstend to be genetically unstable, including polynucleation, and loss and gaining whole or partial chromsomes. Moreover, some undesirable changes, such as loss of some differentiated properties and normal cell-cycle checkpoint controls, immerged inevitably in these cells. Not every cell line was really immortalized as some of them had only overcome senescence to prolong the life span. In addition, human immortalized odontoblast cell line has not been established for the limitation of material source.In order to overcome the shortcomings of these cell lines, human telomerase reverse transcriptase (hTERT) was transfected into primary human odontoblast-like cells. Subsequent activation of telomerase allowed the cells to overcome senescence and crisis and become immortal. So an immortalized human odonto-blast-like cell line was established successfully. As the mechanism of immortalization by activation of telomerase through hTERT also exists in human embroynic stem cells, hTERT-hOd-1 maintained more biological and functional characteristics of human native odontoblasts compared with transformed cell line immortalized with DNA tumor virus. +一, Primary culture of human odontoblast-like cellsHuman deciduous molar tooth germs were isolated from an 8-month-old, stillborn female fetus. The dental papillae were mechanically removed and the cells were prepared using tissue explant culture technique. Primary cells were seeded for plate-cloning and cultured in mineralizing medium. Three cell clones, which took on similar morphology to odontoblasts, were selected using filter paper and expanded in culture. The cells from clone a took on fibroblast-like shape with single and long cellular process, without polarized nucleus. They expressed dentin matrix protein 1(DMP1) and dentin sialophosphoprotein (DSPP), a specific marker of odontoblast, at mRNA level, and showed high alkaline phosphatase (ALP) activity. Under the induction of mineralizingmedium, the cells could form mineralized nodules in vitro. All the evidence suggests that the primary cultured cells be human odontoblast-like cells. +二, Establishment of immortalized human odontoblast-like cell linePrimary human odontoblast-like cells were transfected with eukaryotic expressing plasmid pCIneo-hTERT encoding hTERT and neo genes. After selection with G418 for a month, five anti-G418 cell clones were selected using filter paper and expanded for continuous culture. After stable transfection, hTERT was expressed at mRNA and protein level and telomerase activity turned to be positive in these transfected cells. Cells derived from clone 5b has been maintained in culture for about 6 months, up to 56 passages till now, which was designated as hTERT-immortalized human odontoblast-like cell line (hTERT-hOd-1). +三 Biological characteristics of the cell line hTERT-hOd-1During continuous culture, h.TERT-hOd-1 was fi...
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