体外诱导人牙龈成纤维细胞多向分化潜能的实验研究

Study on differentiation pluripotency of human gingival fibroblasts induced in vitro

目的探索人牙龈成纤维细胞(h GFs)是否具有多向分化潜能,为组织工程学提供新的细胞来源。方法经志愿者知情同意后收集健康牙龈组织,组织块法培养h GFs。取第3代h GFs进行成骨、成软骨和成脂诱导,未分化诱导的细胞为对照组。分别用碱性磷酸酶(ALP)染色和茜素红染色、阿利新蓝染色、油红O染色检测细胞成骨、成软骨和成脂能力。实时定量聚合酶链反应(PCR)检测细胞中成骨分化标志基因ALP、runt相关转录因子2(Runx2)、成软骨分化标志基因聚集蛋白聚糖(AGR)和成脂分化标志基因过氧化物酶体增殖物激活受体γ2(PPARγ2)的表达。结果成骨诱导组培养至7 d时ALP染色可见大量的蓝紫色沉淀,28 d时细胞周围有大量红染的钙结节沉积,而对照组无钙结节,培养至14 d细胞中ALP和Runx2表达明显高于对照组(P<0.01)。14 d时成软骨诱导组阿利新蓝阳性,成脂诱导组可见红色的脂肪滴,而对照组2种染色为阴性,AGR、PPARγ2表达均明显高于对照组(P<0.01)。结论 h GFs具有成骨、成软骨和成脂分化的多向分化潜能。

Objective To investigate the pluripotency of human gingival fibroblasts（h GFs）, and provide a novel cellsource for tissue engineering. Methods With informed consent from volunteers, fresh and healthy gingiva were collected.The h GFs were obtained from the gingiva by tissue culture. The third passage of h GFs was cultured in osteogenic medium,chondrogenic medium and adipogenic medium. Cells without differentiation were taken as control. Cells were examined by al-kaline phosphatase（ALP） staining, Alizarin red staining, Alcian blue staining and oil red O staining for detecting of the abili-ty of differentiation pluripotency. Real-time polymerase chain reaction was applied to examine the expression of osteogenicmarker genes ALP, runt-related transcript factor 2（Runx2）, chondrogenic marker aggrecan（AGR） and adipogenic markerperoxisome proliferator-activated receptor gamma 2（PPARγ2）. Results The h GFs cultured in osteogenic medium showedmassive violet deposit at day 7 and calcium nodulus at day 28, meanwhile, the expressions of ALP and Runx2 were higherthan those of control（P〈0.01）. In chondrogenic group cells were found blue deposit at day 14. In adipogenic group lipid-filled droplets stained with oil red O were found in cells at day 14. However, h GFs in control group had no any positive stain-ing. Furthermore, expressions of AGR and PPARγ2 were significantly higher than those of control（P〈0.01）. Conclusion Human gingival fibroblasts have the pluripotency of osteogenic, adipogenic and chondrogenic differentiation.