Jie Zhou1, Ting Liu, Hong Zheng, Jin-lin Song, Feng Deng. 1. Department of Orthodontics, The Affiliated Hospital of Stomatology, Chongqing Medical University & Chongqing Research Center for Oral Diseases and Biomedical Science, Chongqing 401147, China. soongjl@163.com
Abstract
OBJECTIVE: To construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research. METHODS: The rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test. RESULTS: Morphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice. CONCLUSIONS: The rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.
OBJECTIVE: To construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research. METHODS: The rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test. RESULTS: Morphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice. CONCLUSIONS: The rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.