Abstract:

Objective: To investigate the effects of cathepsin K (Ctsk) on the proliferation, migration, and differentiation of jaw bone marrow-derived mesenchymal stem cells (JBMMSCs) in mice. Methods: Mouse JBMMSCs were cultured and identified in vitro. Third-passage (P3) cells were subjected to osteogenic, chondrogenic, and adipogenic tri-lineage induction. The expression of Ctsk was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Small interfering RNA (siRNA) transfection was used to knockdown Ctsk expression in JBMMSCs, with knockdown efficiency verified by RT-qPCR and Western blotting. Cell proliferation was assessed using the CCK8 assay, and cell migration was evaluated via scratch wound healing assay. The expression of key genes during tri-lineage differentiation was detected by RT-qPCR. Alkaline phosphatase (ALP) staining was used to assess osteogenic mineralization, Alcian blue staining was applied to evaluate cartilage matrix secretion, and oil red O staining was employed to examine lipid droplet formation. Results: Ctsk expression increased after osteogenic and chondrogenic induction, but decreased after adipogenic induction. Following Ctsk knockdown, cellular migration, osteogenesis, and chondrogenesis were attenuated, but proliferation and adipogenic ability were enhanced. Conclusion: Ctsk promotes the migration, osteogenesis, and chondrogenesis in mouse JBMMSCs, but inhibits their proliferation and adipogenic differentiation.

Key words: jaw bone marrow-derived mesenchymal stem cells, cathepsin K, proliferation, migration, differentiation

摘要：

目的：探究组织蛋白酶K（cathepsin K，Ctsk）对小鼠颌骨骨髓间充质干细胞（jaw bone marrow-derived mesenchymal stem cells，JBMMSCs）增殖、迁移及分化的影响。方法：体外培养并鉴定小鼠JBMMSCs，取第三代（P3）细胞进行成骨、成软骨和成脂三向诱导分化，采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测Ctsk的表达变化；通过小干扰RNA（small interfering RNA，siRNA）转染技术敲降JBMMSCs中Ctsk的表达，并利用RT-qPCR及蛋白质印迹法验证敲降效率；采用CCK8法检测细胞增殖能力，划痕实验检测细胞迁移能力；RT-qPCR检测三向分化过程中关键基因的表达；碱性磷酸酶（alkaline phosphatase，ALP）染色评估JBMMSCs的成骨矿化情况；阿利新蓝染色评估软骨基质分泌；油红O染色评估脂滴形成情况。结果：成骨及成软骨诱导后，Ctsk表达增加，成脂诱导后，Ctsk表达下降；敲降Ctsk表达后，细胞的迁移、成骨及成软骨能力减弱，但增殖和成脂能力增强。结论：Ctsk能促进小鼠JBMMSCs迁移、成骨及成软骨分化，但会抑制其增殖及成脂分化。

关键词: 颌骨骨髓间充质干细胞, 组织蛋白酶K, 增殖, 迁移, 分化