ヒト歯髄由来培養細胞のプラスミドｐＭＴ１‐ｎｅｏ遺伝子導入による不死化について

Abstract

A human dental pulp tissue was explanted in culture and the outgrowing cells were transfected with the plasmid, pMT1-neo, which included the early region of SV 40 DNA and the neomycin-resistant gene. The transfected cells were cloned and cultured beyond the period of senescence for the non-transfected HOP cells, over 170 passages (360 population doubling levels and 730 days). The characteristics of the transfected LSC cells and the non-transfected HOP cells were investigated during in vitro aging. The results were as follows: 1. The alkaline phosphatase (ALPase) activity of the HOP cells decreased as the culture passages increased. In contrast, the ALPase activity of the LSC cells did not change during the entire serial culture period. 2. The ALPase inhibitory test indicated that the ALPase in the LSC cells was the bone/liver/kidney type. 3. The addition of 250 micrograms/ml of L-ascorbic acid resulted in an increased collagen synthesis compared with that of 50 micrograms/ml. 4. The growth rate and the ALPase activity of the LSC cells were affected by 1 alpha, 25-dihydroxyvitamin D3, L-ascorbic acid, beta-sodium glycerophosphate, epidermal growth factor or transforming growth factor-beta even after the serial culture. These results suggest that the LSC cells preserve some properties of the dental pulp and may be useful in the future research for exploring the mechanisms of the dental hard tissue formation and for testing the biocompatibility of the dental restorative materials.