Studies for linage of periodontal ligament cells by gene transfection technique.

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To clarify a lineage of cells isolated from periodontal ligament (PDL), the following studies were undertaken. First, we constructed (he experimental protocol for introducing and expression of the foreign genes into primary culture of human PDL cells. We constructed the plasmid vector which have human telomerase gene at downstream for cytomegarovirus immediate early enhancer and promoter, and introduced it to the primary cell culture which were established by the outgrowth method from human PDL. The expression of telomerase gene in cloned cells selected by an antibiotic marker was confirmed by RT-PCR. Secondly, expression of alkaline phosphatase (ALP) which were known to be a classical marker for cells in osteoblastic lineage were analyzed in single cell derived-clones form human PDL cells. The cloned cells showed same morphology, however expressed different levels of ALP activities and showed different responses against dexamethasone. The RNA expression levels for ALP gene were almost identical among these clones, suggesting that the different levels for ALP activities were derived from translational and/or transmembrane process of ALP protein. Thirdly, we investigated the gene-expression profiles in different clones for human PDL cells. Total RNAs expressed in two different PDL clones, one is high ALP clone and other is low ALP clone, were analyzed by a microarray assay, and compared with each other in over 1,500 different genes. These PDL clones showed almost same RNA expression profiles and showed slight difference in several specific genes. Finally, we also analyzed the roles of nerve growth factors in mouse PDL clones.