GDF11通过介导PI3K/AKT通路对人牙髓干细胞体外矿化能力的影响研究

The effects of GDF11 on the mineralization of human dental pulp stem cells by PI3K/AKT pathway

目的:探究GDF11在人牙髓干细胞(hDPSCs)矿化过程中的作用及潜在机制。方法:收集拔除的25岁以下患者第三磨牙组织,用成骨培养基诱导培养原代hDPSCs,对传代培养的细胞进行流式细胞术hPDSCs鉴定。用RNA干扰技术对hDPSCs进行生长分化因子11(GDF11)基因敲减实验。免疫荧光染色用于验证hDPSCs中GDF11的表达和分布,商业试剂盒用于检测hDPSCs牙源性分化和矿化能力;qRT-PCR用于检测牙本质相关标志物(牙本质唾液酸磷蛋白(DSPP)、骨钙素(OCN)和Ⅰ型胶原α1链(COL1a1))的表达水平;Western Blot用于检测PI3K/AKT信号通路中相关蛋白分子的蛋白磷酸化蛋白激酶B(p-AKT)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR))表达水平。结果:GDF11在hDPSCs中表达主要分布在细胞质中,敲减GDF11后可减弱牙源性分化和矿化能力(P<0.05),导致牙本质标志物相关基因DSPP、OCN和COL1a1的转录水平显着下调(P<0.05),并下调PI3K/AKT信号通路中相关蛋白分子的蛋白p-AKT和p-mTOR表达(P<0.05)。结论:GDF11通过PI3K/AKT信号通路正向调节h DPSCs的牙源性分化和矿化。

Objective:To explore the role and potential mechanism of GDF11 in the mineralization of human dental pulp stem cells(hDPSCs).Methods:The pulp tissue of the extracted third molar of the subjects under 25 years old were collected and the isolated pulp cells were cultured with osteogenesis induction medium.The subcultured cells were identified to be hPDSCs by flow cytometry.RNA inference technology was used to knockdown GDF11 in hDPSCs.Immunofluorescence staining was used to verify the expression and distribution of GDF11 in hDPSCs.Commercial kits were used to detect the dentin differentiation and mineralization ability of hDPSCs.qRT-PCR was used to detect the expression level of dentin-related markers DSPP,OCN and COL1a1.Western Blot was used to detect the associated-protein levels of p-AKT and p-mTOR in the PI3K/AKT signaling pathway.Results:The expression of GDF11 in hDPSCs was mainly distributed in the cytoplasm.Knockdown of GDF11 decreased the ability of dentin differentiation and mineralization(P<0.05),resulting decrease in the transcription level of dentin marker-related genes DSPP,OCN and COL1a1(P<0.05),and suppressed the expression of related-protein p-AKT and p-mTOR in the PI3K/AKT signaling pathway(P<0.05).Conclusion:GDF11 positively regulates the odontogenic differentiation and mineralization of hDPSCs through PI3K/AKT signaling pathway.