Alternative TitleCharacterization of an oral fibroblast phenotype cultured in oral keratinocyte-conditioned medium
口腔粘膜上皮細胞培養上清で培養した口腔粘膜繊維芽細胞の形質に関する検討
Note (General)Fibroblasts in monoculture grown in serum-containing medium are highly-proliferating. However, data suggested that use of static fibroblasts provides a better model to study biological phenomena than proliferating fibroblasts even when constructing a three-dimensional matrix. Since previous studies have stated keratinocyte-conditioned medium reduced fibroblast proliferation, this study aimed to examine if we can harness the discarded oral keratinocyte-conditioned medium (OK-CM) for oral fibroblasts (OF) culture in a monolayer and to investigate their phenotypic changes by OK-CM from an actively-cycling state to a more static state as well as their behavior. Primary human OK and OF were grown in a chemically-defined, completed EpiLife and DMEM containing 10% calf serum (DMEM-CS), respectively. OK-CM conditioned for 24 hours was obtained from near-confluent OK culture. OF were plated into microplate-wells, and 24 hours later, the medium was replaced with DMEM-CS, serum-free DMEM (SF-DMEM) and OK-CM. OF were cultured up to 96 hours. Proliferation rate and cell cycle profile were analyzed using a MTT assay and a fluorescence-activated cell sorter. To assess the phenotypic changes of OFs cultured in each medium, the activity of senescent-associated β-GAL and the secreted protein levels including Keratinocyte growth factor (KGF), human type I collagen and MMP-1 were determined. The proliferating rate and the proportion of cells in S and G2/M significantly decreased when cells were cultured in OK-CM, indicating the inhibition of cell cycle progression at the G1 phase. In contrast to OFs in SF-DMEM, only a few OFs cultured in OK-CM showed the β-gal activity, suggesting that they had proliferating potential. ELISA assay showed OFs cultured in OK-CM produce KGF and MMP-1 as do OFs grown in DMEM-CS. However, their ability to produce type I collagen significantly decreased compared with OFs in DMEM-CS. Significant OK-CM-mediated OF phenotypic changes suggest the feasibility of those quiescent OFs cultured in OK-CM for reconstruction of a three-dimensional tissue-engineered oral mucosa substitute.
学位の種類: 博士（歯学）. 報告番号: 甲第3816号. 学位記番号: 新大院博（歯）甲第282号. 学位授与年月日: 平成25年9月20日
新大院博（歯）甲第282号
Collection (particular)国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文
Date Accepted (W3CDTF)2015-02-03T05:25:05+09:00
Data Provider (Database)国立国会図書館 : 国立国会図書館デジタルコレクション