炎症细胞因子刺激下胰岛素样生长因子结合蛋白5对牙周膜干细胞成骨分化的积极效应

Effects of insulin-like growth factor-binding protein 5 on osteogenic differentiation of periodontal ligament stem cells stimulated by inflammatory cytokines

背景:既往已有研究证实胰岛素样生长因子结合蛋白5(insulin-like growth factor-binding protein 5,IGFBP5)在牙源性干细胞中高表达,并通过激活多种信号通路发挥作用。然而,在炎症微环境条件下IGFPB5是否参与调控间充质干细胞骨向分化尚不清楚。目的:探讨IGFBP5在炎症微环境下对牙周膜干细胞成骨分化的影响及分子调控机制,为炎症微环境下牙周组织再生寻找治疗靶点。方法:(1)采用酶消化法分离培养人牙周膜干细胞,通过流式细胞术检测其表面抗原标志物;(2)分别以肿瘤坏死因子α、白细胞介素17模拟体内炎症微环境作用,并建立IGFBP5稳定敲低的细胞系,然后进行成骨诱导分化,检测碱性磷酸酶活性、成骨分化相关标志性基因及相关的组蛋白去甲基化酶的表达。该研究的实施符合天津医科大学口腔医院的相关伦理要求。结果与结论:(1)加入肿瘤坏死因子α、白细胞介素17后,牙周膜干细胞成骨能力降低,具体表现为碱性磷酸酶活性及成骨分化相关基因OCN、BSPmRNA表达显著降低;(2)基因沉默IGFBP5进一步加重了肿瘤坏死因子α、白细胞介素17对牙周膜干细胞的成骨抑制作用,表现在碱性磷酸酶活性及成骨分化相关基因OCN、BSP mRNA表达进一步降低;(3)炎症因子刺激及基因沉默IGFBP5同时影响了组蛋白去甲基化酶KDM2A、KDM3B、KDM5B、JMJD6 mRNA的表达;(4)结果提示IGFBP5在炎症微环境下对牙周膜干细胞的成骨分化过程可能起到积极作用。

BACKGROUND: Previous studies have confirmed that insulin-like growth factor-binding protein 5 (IGFBP5) is highly expressed in dental stem cells and exerts roles by activating multiple signaling pathways. However, it is unclear whether IGFPB5 is involved in the regulation of mesenchymal stem cell osteogenic differentiation in inflammatory microenvironment. OBJECTIVE: To study the influence and molecular mechanism of IGFBP5 on osteogenic differentiation of periodontal ligament stem cells under inflammatory microenvironment, and to provide a new therapeutic target for periodontal tissue regeneration in inflammatory microenvironment. METHODS: After periodontal ligament stem cells were isolated and collected, their surface antigen markers were detected by flow cytometry. The stable IGFBP5 knock-down cell lines were established, and were cultured in osteogenic differentiation medium containing tumor necrosis factor α and interleukin 17. We then detected alkaline phosphatase activities and mRNA expression of marker genes relating to osteogenic differentiation and histone methyltransferase. The study protocol was implemented in line with the ethic requirements of Tianjin Medical University Hospital of Stomatology. RESULTS AND CONCLUSION: (1) The osteogenic capacity of periodontal ligament stem cells decreased under stimulation of tumor necrosis factor α and interleukin 17. Compared with the blank control group, the activity of alkaline phosphatase and the expressions of osteogenic differentiation related genes OCN and BSP were significantly reduced after osteoblastic induction. (2) Gene silencing of IGFBP5 allowed tumor necrosis factor α and interleukin 17 to further inhibit the osteogenesis of periodontal ligament stem cells, characterized by decreased alkaline phosphatase activity and further decreased expression of osteoblastic differentiation related genes OCN and BSP. (3) Inflammatory factor stimulation and gene silencing of IGFBP5 also affected the mRNA expression of histone demethylases KDM2A, KDM3B, KDM5B and JMJD6. To conclude, IGFBP5 may play a positive role in the osteogenic differentiation of periodontal ligament stem cells.