CAJ | 학술논문

目的观察不同浓度的血小板衍生因子BB(PDGFBB)和碱性成纤维细胞生长因子(bFGF)对人肌腱细胞的表型和表型标志物基因表达的影响.方法存添加1％胎牛血清(FBS)的α-MEM培养基中,加入PDGFBB(50g/L)、bFGF(50 μg/L)以及50 μg/L PDGFBB+50 μg/L bFGF,使用10％的FBS为阳性对照,用天狼星红染色细胞观察细胞表型变化,在细胞培养第14天提取各组细胞mRNA,采用实时定量逆转录聚合酶链反应(RT-qPCR)的方法测定人肌腱细胞表型及分化标志物基因表达.结果在添加了1％ FBS条件下,各实验组及对照组细胞形态均未发现表型变异,与阳性对照组比较,1％ FBS+50 μg/L PDGFBB+50 μg/L bF(GF组的细胞形态呈现出较弱分化,产生胶原较少,胶原纤维的排列杂乱没有阳性对照那样的浓密有序及平型排列的胶原纤维形态.与阳性对照组比较,1％ FBS+50 μg/L PDGFBB +50 μg/L bFGF组的肌腱细胞表型标志物的基因表达的比值明显下调[Scleraxis(Scx)和Decorin (Dcn)0.33,t=374.055;Tenomodulin(Tnmd)0.003,t=28 199.418;Ⅰ型胶原(Col Ⅰ)0.57,t=164.195; Dcn 0.26,t=177.520],差异有统计学意义(P＜0.05).结论使用1％ FBS的α-MEM培养基中添加50 μg/LPDGFBB+50 μg/L bFGF可维持人肌腱细胞表型,胶原纤维产生较添加10％ FBS培养的细胞明显减少,同时下调肌腱细胞的表型及分化标志物的mRNA表达.
목적 관찰불동농도적혈소판연생인자BB(PDGFBB)화감성성섬유세포생장인자(bFGF)대인기건세포적표형화표형표지물기인표체적영향.방법 존첨가1％태우혈청(FBS)적α-MEM배양기중,가입PDGFBB(50g/L)、bFGF(50 μg/L)이급50 μg/L PDGFBB+50 μg/L bFGF,사용10％적FBS위양성대조,용천랑성홍염색세포관찰세포표형변화,재세포배양제14천제취각조세포mRNA,채용실시정량역전록취합매련반응(RT-qPCR)적방법측정인기건세포표형급분화표지물기인표체.결과 재첨가료1％ FBS조건하,각실험조급대조조세포형태균미발현표형변이,여양성대조조비교,1％ FBS+50 μg/L PDGFBB+50 μg/L bF(GF조적세포형태정현출교약분화,산생효원교소,효원섬유적배렬잡란몰유양성대조나양적농밀유서급평형배렬적효원섬유형태.여양성대조조비교,1％ FBS+50 μg/L PDGFBB +50 μg/L bFGF조적기건세포표형표지물적기인표체적비치명현하조[Scleraxis(Scx)화Decorin (Dcn)0.33,t=374.055;Tenomodulin(Tnmd)0.003,t=28 199.418;Ⅰ형효원(Col Ⅰ)0.57,t=164.195; Dcn 0.26,t=177.520],차이유통계학의의(P＜0.05).결론 사용1％ FBS적α-MEM배양기중첨가50 μg/LPDGFBB+50 μg/L bFGF가유지인기건세포표형,효원섬유산생교첨가10％ FBS배양적세포명현감소,동시하조기건세포적표형급분화표지물적mRNA표체.
Objective To evaluate the the changes of tenocyte phenotype and the expression of the phenotypic markers by adding platelet derived growth factor BB (PDGFBB) and basic fibroblast growth factor(bFGF) to human tenocyte in vitro culture.Methods Human tenocytes were cultured in α-MEM medium by adding FBS at the concentration of 1％ and supplementing either/both PDGFBB and bFGF.Sirius red staining was employed to evaluate the characteristics of the tenocytes cultured.Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) technique was used to detect the mRNA expression of the tenocyte phenotypic and differentiation markers.Results The tenocytes cultured for 14 days with 1％ FBS,50 μg/L PDGFBB + 50 μg/L bFGF showed less prominent collagen synthesis in comparison with those cultured in 10％ FBS.The tenocytes cultured in the treated group also showed down-regulated mRNA expression of those markers [Scleraxis (Scx) 0.33,t =374.055 ; Tenomodulin (Tnmd) 0.003,t =28 199.418; Collagen type Ⅰ (Col Ⅰ) 0.57,t =164.195 ; Decorin (Den) 0.26,t =177.52).Conclusion These findings suggest that human tenocytes could not only maintain their phenotype in the culture media with low concentration of FBS,but also keep the cells at less differentiated state,having this approach a suitable one for tendon tissue engineering.