Extension human oral keratinocytes life-span by introduction of telomerase

Objectives: To introduce human telomerase reverse transcriptase (hTRT) into normal human oral keratinocytes(OKC) to extend their life-span. Methods: hTRT cDNA was transfected into OKC by pBABE-tert retravirus. Telomerase activity was analysed by telomerase PCR ELISA. Exogenous hTRT was analyzed by RT-PCR. Morphological characteristics, cellular kinetics, chromosome analysis, ©¬-galactosidase staining and tumor formation in nude mice were used to compare transfeced OKC with normal OKC. Results: The life-span of OKC transfeced by pBABE-tert retravirus was doubled to 8-10 passages which was due to the transfection of exogenous hTRT and reconstruction of telomerase activity. Transfeced OKC has normal phenotype and no sign of malignant transformation. Conclusions: Reconstruction of telomerase activity could extended the life-span of normal OKC on a limited scale. This study is of particular importance not only to oral carcinogenesis, pathology and toxicology but also to cell senescence study and tissue engineered oral mucosa fabrication to treat large defects of oral mucosa. Key words Oral Keratinocytes/Epithelial cells Telomerase hTRT/hTERT Gene transfection