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KMID : 0363020020320030603
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Journal of Korean Academy of Periodontology
2002 Volume.32 No. 3 p.603 ~ p.614
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Establishment of Immotalized Human Gingival Fibroblast Cell Lines
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송제봉
김현아/현하나/김은철/유형근/신형식
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Abstract
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Human gingival fibroblasts have proven to useful as a species specific cell culture system in various system on periodontal disease and regeneration. However, their use is limited, since they are hard to obtain and lifespan is short due to replicative senescence. To overcome these disadvantages, we transfected primary human gingival fibroblasts by the E6 and E7 genes of the Human papilloma virus(HPV) 16. The full length of HPV 16 E6 and E7 was cloned from the pBR322 into BamH1 and Sal I of a pBabe vector including hygromycin B resistance. Before pBabeE6/E7 plasmid transfection, peak 8 GFP including G418 resistance was transfected into primary GF to check the transfection efficency. PBabe E6/E7 plasmid was transfected using Lipofectamine plus following manufacter's instruction into primary normal human gingival fibroblasts in 60mm dishes with FBS free DMEM. After 2 days of transfection, the cells were treated with hygromycin for 2 weeks until the transfected control cells died. The resulting hygromycin resistant colonies were pooled, and clonned, and sucessful transfection was established for immortalized gingival fibroblast cell lines.
Immoralized GF cells showed stellate shape, that is similar to that of orange grains, and more rapid growth and higher proliferation than that of primary gingival fibroblasts.
This cell lines overcame crisis and could be cultured over 30 subcultured, could be use for three dimentional culture, epithelial-mesenchymal interaction study.
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KEYWORD
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Gingival Fibroblast, Cell line, Immortalization, HPV, Growth, Proliferation
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