Construction Of Human Oral Epithelial Cell Lines Overexpressing HTERT Gene Mediated By Lentivirus

PURPOSE: Oral mucosal disease occur in the oral mucosa is the general term for all diseases, its wide range of complex etiology and pathogenesis of most diseases is not yet entirely clear. Which originated in the incidence of malignant tumors not only the oral mucosa and affect people’s quality of life has become a common disease, even a serious threat to life and health. Due to normal OMECs vitro difficulties, high cell nutritional requirements and shorter survival time, usually 5-6 subculture began after the death of aging generations, which severely limits our in-depth study of its pathogenesis, therefore, an urgent need for a capable long-term stability passage OMECs lines as in vitro experimental model cells.1.Our research aimed to construct the recombination lentivirus vector pLVX-puro- hTERT and packaged by 293 Tcells. 2.Used the recombination vector to infect primary oral epithelial cells, To establish a stable cell line of oral epithelial cell by transfection with human telomerase reverse transcriptase(hTERT),To explore a new method of establishing a stable and efficient immortalized oral epithelial cell line.METHODS: The total RNA extracted from 293 Tcells and the hTERT gene was amplified in polymerase chain reaction(PCR). Then, we used it to construct the recombination lentivirus vector of p LVX-puro-hTERT. After that, lentivirus particals were successfully packaged and used to infect primary oral epithelial cell. The positive cell clones were selected by puromycin. Finally, we also examined the expression of hTERT by Real-time PCR and Western blot.RESULTS: 1.Amplification results of hTERT gene,1% agarose gel electrophoresis shows 3 KB place a belt of light, in accordance with purpose stripe size.2.pLVX-puro-hTERT digested with NheI and EcoRI,shows 3.3 KB place a belt of light, Preliminary showed that hTERT is successful in slow virus vector pLVX – puro.3.Sequencing results further confirmed that the restructuring slow virus vector pLVX puro- hTERT build is successful.4.The morphology of hTERT OMECs observed by inverted phase contrast microscope are similar to normal OMECs forms, as the polygon, "aura" pattern, the epithelial cells of the typical characteristics.And cells after transfection empty carrier form is irregular, not transfection cells and cells after transfection empty carrier, slow growth after four to five generation gradually died, and transfection of hTERT OMECs still grows fast, stable proliferation.5.The relative expression of hTERT in infected cells measured by qRT-PCR, The expression of hTERTmRNA abundance highly in transfection cells,show significant differenceas compared with normal OMECs(P < 0.01)6. according to the results of Western blot in transfection cells, The hTERT protein is expressed successfully and shows a significant difference with normal cells group.This proved that hTERT gene has been integrated into the target cell’s genome and translated the protein.CONCLUSIONS: This study proved the possibility and superiority that used the gene hTERT medicated by lentivirus to immortalize the oral mucosa epithelial cells. And we might provide a new strategy to explore constructing the immortalized the oral mucosa epithelial cells.