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目的:探讨多能性转录因子Oct4对人牙周膜干细胞(h PDLSCs)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h PDLSCs。RT-PCR检测h PDLSCs矿化诱导分化前后Oct4表达水平;用shRNA慢病毒转染法抑制h PDLSCs内Oct4的表达后,观察h PDLSCs增殖和克隆形成能力的变化;茜素红和油红O染色观察Oct4表达降低对h PDLSCs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:h PDLSCs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,h PDLSCs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Oct4表达后,h PDLSCs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h PDLSCs干性特征中发挥着重要作用。
AIM: To investigate the regulatory effect of pluripotency transcription factor Oct4 on self-renewal ability of human PDLCs. Methods: The h PDLSCs were isolated and purified by limiting dilution method. RT-PCR was used to detect the expression of Oct4 in h PDLSCs induced by mineralization before and after differentiation. The expression of Oct4 in h PDLSCs was inhibited by shRNA lentiviral transfection, and the proliferation and clonogenic ability of h PDLSCs were observed. Alizarin red and oil red O staining The effect of Oct4 expression on the osteogenic and adipogenic differentiation of h PDLSCs in vitro was observed. The expression of osteoblasts and adipogenic differentiation genes were detected by RT-PCR. Results: After PDLSCs were mineralized, the expression level of Oct4 was significantly down-regulated compared with that before differentiation. The proliferation and colony formation rate of h PDLSCs were significantly reduced after Oct4 expression (P <0.05). The formation of calcified nodules and lipid droplets (PPARγ) and osteoblast / odontoblast-related genes (DSPP, ALP, OCN, BSP) were also significantly decreased (P <0.05). CONCLUSION: Inhibition of multipotential transcription factor Oct4 expression can reduce the proliferation, colony formation and multidirectional differentiation of h PDLSCs, suggesting that Oct4 plays an important role in maintaining the dry features of h PDLSCs.