北京大学学报（医学版）2017，Vol.49Issue(1)：16-24,9.DOI:10.3969/j.issn.1671-167X.2017.01.003
不同代次诱导多能干细胞增殖及牙周定向分化的能力
Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages
摘要
Abstract
Objective:To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages,and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity,thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs.Methods:IPSCs derived from human gingival fibroblasts at passages 5,10,15 and 20 were recovered and cultured in vitro.Their morphology and proliferation rates were observed respectively.We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine,then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining,including bone related markers:osteocalcin (OCN),bone sialoprotein (BSP);periodontal ligament related markers:periostin,vimentin;and cementum related markers:cementum attachment protein (CAP),cementum protein 1 (CEMP1).The untreated spontaneous differentiation groups were set as negative controls respectively.Results:iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindlelike shape similar to fibroblasts upon periodontal specific differentiation.All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls.The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5:t =2.125,P =0.003;P10:t =2.246,P =0.021;P15:t =3.754,P =0.004;P20:t =3.933,P =0.002),but no significant difference in their calcium deposition were detected within passages 5,10,15 and 20 (periodontal differentiation:F =2.365,P =0.109;spontaneously differentiation:F =2.901,P =0.067).Periodontal tissue related marker expressions of iPSCs at all passages had also been significandy upgraded under the treatment of GDF-5 (P ＜ 0.05),but still,no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP:F =0.926 7,P=0.450;vimentin:F=0.917 1,P=0.455;CEMP1:F=2.129,P=0.1367).Conclusion:Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs,as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging.Therefore,iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.关键词
多能干细胞/细胞代次/牙周定向分化/细胞增殖Key words
Multipotent stem cells/Cell passages/Periodontal specific differentiation/Cell proliferation分类
医药卫生引用本文复制引用
李静文,殷晓晖,栾庆先..不同代次诱导多能干细胞增殖及牙周定向分化的能力[J].北京大学学报（医学版）,2017,49(1):16-24,9.基金项目
国家自然科学基金(81470739)资助 Supported by the National Natural Science Foundation of China (81470739) （81470739）
