组合应用血小板衍生因子BB和碱性成纤维细胞生长因子对人肌腱细胞表型和表型及分化标志物mRNA表达的影响

Effects of platelet derived growth factor BB and basic fibroblast growth factor on phenotype of tenocytes and the mRNA expression of tenocyte phenotypic and differentiation markers

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。方法在添加1％胎牛血清（FBS）的d—MEM培养基中，加入PDGFBB（50g／L）、bFGF（50μg/L）以及50μg/LPDGFBB＋50μg/LbFGF，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＋50μg/LPDGF。B-4-50μg／LbFGF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＋50μg／LPDGF8B＋50μg／LbFGF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=28199．418；I型胶原（ColI）0．57，f=164．195；Dcn0．26，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50μg/LPDGFBB＋50μg／LbFGF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。

Objective To evaluate the ihe changes of tenocyte phenotype and the expression of the phenotypic markers by adding platelet derived growth factor BB （ PDGFBB ） and basic fibroblast growth fac- tor（bFGF） to human tenocyte in vitro culture. Methods Human tenocytes were cultured in α-MEM medi- um 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 tran- scriptase-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, 50μg/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. S7 ,t = 164. 195 ; Decorin （Dcn） 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.