南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (10): 1439-1447.doi: 10.12122/j.issn.1673-4254.2021.10.01

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活化的Mir-30a-wnt/β-catenin信号轴通过上调组织蛋白酶K的表达促进牙周膜干细胞向成牙骨质细胞分化

刘 芬，周志斐，薛 洋，朱 斌，吴补领，陈发明   

  1. 西北妇女儿童医院口腔科，陕西 西安 710000；第四军医大学口腔医院牙周病科，颌面外科，陕西 西安 710000；南方医科大学深圳医院颌面外科，广东 深圳 518000；西藏军区总医院口腔科，西藏 拉萨 850000； 南方医科大学深圳口腔医院（坪山），广东 深圳 518000
  • 出版日期:2021-10-20 发布日期:2021-11-11

Activation of mir-30a-wnt/β-catenin signaling pathway upregulates cathepsin K expression to promote cementogenic differentiation of periodontal ligament stem cells

LIU Fen, ZHOU Zhifei, XUE Yang, ZHU Bin, WU Buling, CHEN Faming   

  • Online:2021-10-20 Published:2021-11-11

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摘要： 目的 探讨牙周膜干细胞（PDLSC）成牙骨质向分化过程中mir-30a-wnt/β-catenin信号轴调控组织蛋白酶K（CTSK）表达的功能作用。方法 极限稀释法分离培养PDLSC，釉基质蛋白衍生物（EMD）诱导细胞成牙骨质向分化。在细胞分化过程中，通过microRNA芯片筛选差异表达的microRNA。首先，给予不同分组细胞EMD诱导和/或抑制microRNA表达等处理，无干预组细胞作为对照。利用qPCR和Western blot技术首先验证差异表达的microRNA是否能够调控CTSK的表达。之后，将细胞膜片复合陶瓷支架材料植入裸鼠皮下，利用免疫组织化学方法观察成牙骨质细胞标记蛋白CAP和CEMP-1的表达变化，明确受microRNA 调控的 CTSK 表达变化是否参与了 PDLSC 成牙骨质分化。在此基础上，从蛋白学水平观察wnt信号通路在microRNA调节CTSK表达过程中是否发挥了相应功效。结果 EMD诱导PDLSC成牙骨质向分化过程中，多种microRNA表达发生变化，其中miR-30a表达出现特异性上调（P<0.05）。表达上调的miR-30a进一步调节CTSK的表达（P<0.05），促进PDLSC异位形成类牙骨质样结构，高表达成牙骨质细胞标记蛋白CAP和CEMP-1。抑制wnt/β-catenin信号通路，miR-30a对CTSK的表达调控作用出现相应减弱。结论 EMD可能通过上调miR-30a的表达激活wnt/β-catenin信号通路从而经mir-30a-wnt/β-catenin信号轴调控CTSK诱导PDLSC向成牙骨质细胞方向分化。

关键词: 牙周膜干细胞, 组织蛋白酶K, 成牙骨质向分化, 微小RNA, wnt信号通路

Abstract: Objective To explore the role of cathepsin K (CTSK) regulated by mir-30a-wnt/β-catenin signaling pathway in cementogenic differentiation of periodontal ligament stem cells (PDLSCs). Methods Human PDLSCs isolated by limiting dilution culture were induced by enamel matrix protein derivative (EMD) for differentiation into cementoblast-like cells. MicroRNA chip technique was employed to screen the differentially expressed microRNAs in the cells during induced differentiation. The effect of inhibiting miR-30a on CTSK expression in the induced cells was examined using RT-PCR and Western blotting. Ceramic scaffolds coated with PDLSCs treated with EMD and transfected with the miR-30a inhibitor or a lentiviral vector for CTSK overexpression were prepared and implanted subcutaneously in nude mice, and 8 weeks later the cellular expressions of cementoblast markers CAP and CEMP-1 were detected with immunohistochemistry to verify whether CTSK participate in cementogenic differentiation of PDLSCs. The role of wnt signaling pathway in miR-30a-mediated regulation of CTSK expression was explored by examining CTSK protein expressions after blocking wnt signaling in PDLSCs. Results In PDLSCs with EMD-induced differentiation into cementoblast-like cells, multiple microRNAs exhibited differential expressions; and among them, miR-30a was specifically and significantly up-regulated (P<0.05). Up-regulation of miR-30a obviously increased the expression of CTSK (P<0.05) and promoted PDLSCs to form cementum-like tissues with high expressions of CAP and CEMP-1. The regulatory effect of miR-30a on CTSK expression was obviously attenuated after inhibiting wnt/β-catenin signaling pathway. Conclusion EMD induces cementogenic differentiation of PDLSCs possibly by up-regulating the expression of miR-30a, which further activates the wnt/β-catenin signaling pathway to enhance the expression of CTSK.

Key words: periodontal ligament stem cells, cathepsin K, cementogenic differentiation, microRNA, wnt signaling pathway