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

doi:10.3969/j.issn.2095-4344.1823

Abstract

Abstract:

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.

Key words: insulin-like growth factor binding protein 5, periodontal ligament stem cells, osteogenic differentiation, inflammatory microenvironment, tumor necrosis factor α, interleukin 17, National Natural Science Foundation of China

CLC Number:

Liu Dayong, Wang Yingchengyao, Sun Ruixin, Liu Fan, Jia Zhi. Effects of insulin-like growth factor-binding protein 5 on osteogenic differentiation of periodontal ligament stem cells stimulated by inflammatory cytokines[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5256-5262.

Figures/Tables 3

2.2 基因沉默IGFBP5对牙周膜干细胞在肿瘤坏死因子α、白细胞介素17刺激下成骨分化的影响 2.2.1 碱性磷酸酶的表达在成骨诱导第7天，对正常牙周膜干细胞、基因沉默IGFBP5的牙周膜干细胞进行碱性磷酸酶染色。与成骨分化组相比，加入肿瘤坏死因子α、白细胞介素17分别处理后碱性磷酸酶表达降低，而基因沉默IGFBP5后碱性磷酸酶表达进一步降低[17]，见图2。在成骨诱导第5天，检测碱性磷酸酶活性这一早期成骨分化指标。成骨诱导条件下，基因沉默IGFBP5使得碱性磷酸酶活性显著降低。与成骨分化组相比，肿瘤坏死因子α组、白细胞介素17组碱性磷酸酶活性均有显著降低，基因沉默IGFBP5使得碱磷酶活性进一步减低。各组数据差异均有显著性意义(P < 0.05)，见图3。实验结果证明，加入肿瘤坏死因子α、白细胞介素17炎症刺激后牙周膜干细胞的碱性磷酸酶表达明显降低，基因沉默IGFBP5可以进一步抑制炎症微环境下牙周膜干细胞的碱性磷酸酶表达。 2.2.2 成骨相关分子的表达成骨诱导第7天，采用RT-PCR法检测成骨相关分子OCN、BSP mRNA表达。与正常的牙周膜干细胞相比，基因沉默IGFBP5的牙周膜干细胞成骨相关分子OCN、BSP表达降低；加入肿瘤坏死因子α后，基因沉默IGFBP5的牙周膜干细胞成骨相关基因表达均显著降低，加入白细胞介素7后，基因沉默IGFBP5的牙周膜干细胞成骨分子OCN表达降低，而BSP表达无显著变化，见图4。 2.2.3 组蛋白去甲基化酶KDM2A、KDM3B、KDM5B及JMJD6 mRNA表达见图4。与成骨分化组相比，加入炎症因子肿瘤坏死因子α、白细胞介素17刺激后，KDM2A mRNA表达水平均显著升高。成骨分化后，与正常牙周膜干细胞相比，基因沉默IGFBP5牙周膜干细胞KDM2A mRNA的表达变化差异无显著性意义。在肿瘤坏死因子α组中观察到，与正常牙周膜干细胞相比，基因沉默IGFBP5造成KDM2A mRNA表达显著降低。在白细胞介素17组中观察到，与正常牙周膜干细胞相比，基因沉默IGFBP5使得KDM2A mRNA表达显著升高。与成骨分化组相比，加入炎症因子肿瘤坏死因子α、白细胞介素17刺激后，KDM3B mRNA表达水平均显著降低。成骨诱导条件下，与正常牙周膜干细胞比较，基因沉默IGFBP5牙周膜干细胞KDM3B mRNA的表达显著降低；在肿瘤坏死因子α组中观察到，与正常牙周膜干细胞相比，基因沉默IGFBP5使得KDM3B mRNA表达进一步减低。在白细胞介素17组中观察到，与正常牙周膜干细胞组相比，基因沉默IGFBP5使得KDM3B mRNA表达水平升高。与成骨分化组相比，加入炎症因子肿瘤坏死因子α、白细胞介素17刺激后，KDM5B mRNA表达水平均显著升高。成骨诱导条件下，与正常牙周膜干细胞比较，基因沉默IGFBP5后KDM5B mRNA的表达降低。在肿瘤坏死因子α组中观察到，与正常牙周膜干细胞相比，基因沉默IGFBP5使得KDM5B mRNA的表达水平进一步升高。在白细胞介素17组中观察到，与正常牙周膜干细胞相比，基因沉默IGFBP5后KDM5B mRNA表达水平略有降低。成骨诱导条件下，与正常牙周膜干细胞比较，基因沉默IGFBP5后JMJD6 mRNA的表达水平升高。成骨诱导条件下，加入肿瘤坏死因子α处理后，与正常牙周膜干细胞相比，基因沉默IGFBP5使得JMJD6 mRNA的表达水平降低。加入白细胞介素17处理后，与正常牙周膜干细胞相比，基因沉默IGFBP5后JMJD6 mRNA的表达水平降低。上述实验证明，肿瘤坏死因子α、白细胞介素17处理后可显著抑制牙周膜干细胞及基因沉默IGFBP5牙周膜干细胞中成骨分化相关分子OCN，BSP mRNA的表达，同时对基因沉默IGFBP5牙周膜干细胞中组蛋白去甲基化酶KDM2A、KDM3B、KDM5B及JMJD6 mRNA的表达均有影响。"

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