多能性转录因子Oct4对人牙周膜干细胞自我更新能力的调控作用

摘要

AIM:To investigate the roles of Octamer binding transcription factor-4 (Oct4) on the self-renewal capacity of human periodontal ligament stem cells (hPDLSCs) in vitro.METHODS:hPDLSCs were isolated from human healthy periodontal tissues.Oct4 expression in hPDLSCs was detected by Real-time PCR after osteogenic induction.Then the proliferation,colony formation,adipogenic and osteogenic differentiation were evaluated after sliencing Oct4 gene of hPDLSCs.Meanwhile,the adipogenic and osteogenic related genes were assessed by Real-time PCR.RESULTS:Oct4 expression of hPDLSCs was down-regulated after osteogenic induction.After silencing Oct4 gene expression the proliferation,colony formation ability of hPDLSCs were inhibited.The mineral nodule and lipid droplet formation of hPDLSCs were also decreased significantly.Furthermore,the gene expression of PPARγ and DSPP,ALP,OCN and BSP were notably reduced.CONCLUSION:Oct4 plays an important role in maintaining the stem cell characteristics of hPDLSCs.%目的:探讨多能性转录因子Oct4对人牙周膜干细胞(hPDLSCs)自我更新能力的调控作用.方法:采用有限稀释法进行细胞克隆纯化培养获得hPDLSCs.RT-PCR检测hPDLSCs矿化诱导分化前后Oct4表达水平;用shRNA慢病毒转染法抑制hPDLSCs内Oct4的表达后,观察hPDLSCs增殖和克隆形成能力的变化;茜素红和油红0染色观察Oct4表达降低对hPDLSCs体外成骨、成脂分化能力的影响,同时用RT-PCR检测成骨、成脂分化相关基因表达水平的变化.结果:hPDLSCs矿化诱导后,Oct4的表达水平较分化前显著下调;抑制Oct4表达后,hPDLSCs的增殖能力、克隆形成率明显降低(P＜0.05);钙化结节和脂滴的形成量也明显减少,成脂基因(PPARγ)和各成骨/成牙本质相关基因(DSPP、ALP、OCN、BSP)的表达水平均显著下调(P＜0.05).结论:抑制多能性转录因子0ct4表达后,hPDLSCs的增殖、克隆形成以及多向分化能力均可降低,提示Oct4在维持hPDLSCs干性特征中发挥着重要作用.