组织蛋白酶K对骨膜间充质细胞成骨分化影响的实验研究

doi:10.12439/kqhm.1005-4979.2026.02.004

摘要/Abstract

摘要：

目的： 探究组织蛋白酶K（cathepsin K，Ctsk）对小鼠骨膜间充质细胞增殖、迁移及成骨分化的影响。方法： 提取小鼠原代骨膜间充质细胞并进行体外培养，通过流式细胞术检测其表面标志物。经0、3、7、14、21 d成骨诱导后，采用实时定量聚合酶链反应（real‐time quantitative polymerase chain reaction，RT‐qPCR）检测Ctsk、Runx2及ALP的表达情况。应用小干扰RNA（small interfering RNA，siRNA）转染骨膜间充质细胞，敲降Ctsk并通过RT‐qPCR检测敲降效率，并检测成骨诱导第3、7、14天时Ctsk及各成骨标志物（Runx2、ALP、BMP2、Sp7、COL1A1）的表达水平变化。通过CCK-8、EdU染色评估细胞增殖能力，Transwell实验和细胞划痕实验检测其迁移能力，应用碱性磷酸酶（alkaline phosphatase，ALP）染色和茜素红S（Alizarin red S，ARS）染色评估敲降Ctsk对其成骨分化能力的影响。结果： 所获细胞高表达CD90.2（92.5%）、CD200（98.8%）、SCA1（92.0%），低表达CD34（2.5%）、CD45（3.2%）、CD105（5.0%）。成骨诱导后，骨膜间充质细胞中Ctsk、ALP及Runx2表达显著上调。转染siRNA后Ctsk敲降效率达62%；敲降Ctsk后，成骨诱导使成骨相关标志物Runx2、ALP、BMP2、Sp7、COL1A1表达下降，细胞增殖能力下降，迁移能力增强，ALP染色及ARS染色明显变浅，矿化结节形成减少。结论： Ctsk可促进骨膜间充质细胞增殖和成骨分化，并抑制其迁移。

关键词: 组织蛋白酶K, 骨膜间充质细胞, 成骨分化

Abstract:

Objective: To investigate the effects of cathepsin K (Ctsk) on the proliferation, migration, and osteogenic differentiation of murine periosteal mesenchymal cells. Methods: Primary periosteal mesenchymal cells were isolated from mice and cultured in vitro, and their surface markers were characterized by flow cytometry. Following osteogenic induction for 0, 3, 7, 14, and 21 days, the expression levels of Ctsk, Runx2, and ALP were evaluated using real‑time quantitative polymerase chain reaction (RT‑qPCR). Ctsk expression was knocked down by transfecting periosteal mesenchymal cells with small interfering RNA (siRNA), and knockdown efficiency was confirmed by RT‑qPCR. The expression of Ctsk and osteogenic markers (Runx2, ALP, BMP2, Sp7, COL1A1) was then examined on days 3, 7, and 14 of osteogenic induction. Cell proliferation was assessed using CCK-8 and EdU staining assays, while cell migration was evaluated by Transwell and wound‑healing assays. The impact of Ctsk knockdown on osteogenic differentiation was determined by alkaline phosphatase (ALP) staining and Alizarin red S (ARS) staining. Results: The isolated cells highly expressed CD90.2 (92.5%), CD200 (98.8%), and SCA1 (92.0%), while showing low expression of CD34 (2.5%), CD45 (3.2%), and CD105 (5.0%). Following osteogenic induction, the expression of Ctsk, ALP, and Runx2 in periosteal mesenchymal cells was significantly upregulated. After siRNA transfection, the Ctsk knockdown efficiency reached 62%. Knockdown of Ctsk resulted in decreased expression of osteogenic markers (Runx2, ALP, BMP2, Sp7, COL1A1), reduced cell proliferation capacity, enhanced migration ability, significantly lighter ALP and ARS staining, and fewer mineralized nodules formed after osteogenic induction. Conclusion: Ctsk promotes proliferation and osteogenic differentiation of periosteal mesenchymal cells while inhibiting their migration.

Key words: cathepsin K, periosteal mesenchymal cells, osteogenic differentiation

中图分类号:

曹熔锴, 王佐林. 组织蛋白酶K对骨膜间充质细胞成骨分化影响的实验研究[J]. 《口腔颌面外科杂志》, 2026, 36(2): 102-111.

CAO Rongkai, WANG Zuolin. Effects of cathepsin K on the osteogenic differentiation of periosteal mesenchymal cells in vitro[J]. 《Journal of Oral and Maxillofacial Surgery》, 2026, 36(2): 102-111.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2026/V36/I2/102

图/表 13

图1 小鼠骨膜间充质细胞的提取

Figure 1 Isolation of mouse periosteal mesenchymal cells

表1 RT-qPCR引物序列

Table 1 Primer sequences for RT-qPCR

基因	正向引物序列(5'-3')	反向引物序列(5'-3')
GAPDH	TGGAAAGCTGTGGCGTGATG	TACTTGGCAGGTTTCTCCAGG
Ctsk	AATTATGGCTGTGGAGGCGG	TGCATTTAGCTGCCTTTGCC
Runx2	AAGGCACAGACAGAAGCTTGA	AGGACTTGGTGCAGAGTTCAG
ALP	TGAGCGACACGGACAAGAAG	CTGGTAGTTGTTGTGAGCGTAATC
COL1A1	TAAGGGTCCCCAATGGTGAGA	GGGTCCCTCGACTCCTACAT
BMP2	GGGACCCGCTGTCTTCTAGT	TCAACTCAAATTCGCTGAGGAC
Sp7	ATGGCGTCCTCTCTGCTTG	TGAAAGGTCAGCGTATGGCTT

图2 小鼠骨膜间充质细胞的体外培养（×40） A.原代骨膜间充质细胞自骨块爬出；B. P1代骨膜间充质细胞；C. P2代骨膜间充质细胞。

Figure 2 In vitro culture of mouse periosteal mesenchymal cells（×40）

图3 流式细胞术鉴定小鼠骨膜间充质细胞表面标志物 A.流式细胞仪细胞筛选过程；B—G. CD34^-、CD45^-、CD105^-、CD90.2⁺、CD200⁺、SCA1⁺的表达情况。

Figure 3 Identification of surface markers of mouse periosteal mesenchymal cells using flow cytometry

图4 骨膜间充质细胞成骨诱导后Ctsk、Runx2及ALP表达量的变化成骨诱导0~21 d时，Ctsk、Runx2、ALP基因相对表达量，*表示P<0.05，与0 d时比较。

Figure 4 Changes in expression of Ctsk, Runx2, and ALP after osteogenic induction of periosteal mesenchymal cells

图5 Ctsk敲降效率 **表示P<0.01，与对照组比较。

Figure 5 Knockdown efficiency of Ctsk

图6 敲降Ctsk后成骨诱导3、7、14 d时Ctsk及成骨标志物表达量的变化 A.成骨诱导3 d时，RT-qPCR检测成骨标志物表达量的变化；B.成骨诱导7 d时，RT-qPCR检测成骨标志物表达量的变化；C.成骨诱导14 d时，RT-qPCR检测成骨标志物表达量的变化。*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 6 Changes in expression of Ctsk and osteogenic markers on days 3, 7, and 14 of osteogenic induction after Ctsk knockdown

图7 敲降组与对照组CCK-8实验结果 *表示P<0.05，与对照组比较。

Figure 7 CCK-8 assay results in knockdown and control groups

图8 敲降组与对照组EdU染色结果 A. EdU染色实验结果（×100）；B. EdU染色结果统计分析，*表示P<0.05，与对照组比较。

Figure 8 EdU staining results in knockdown and control groups

图9 敲降组与对照组细胞划痕实验结果 A.对照组与敲降组划痕实验结果（×40）；B. 24 h划痕实验迁移细胞计数；C. 48 h划痕实验迁移细胞计数。*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 9 Wound-healing assay results in knockdown and control groups

图10 敲降组与对照组细胞Transwell实验结果 A.敲降组与对照组Transwell实验结果（×100）；B. Transwell实验迁移细胞计数；C.对照组与敲降组的OD值。**表示P<0.01，与对照组比较。

Figure 10 Transwell assay results in knockdown and control groups

图11 敲降组与对照组ALP染色结果（×40） A.对照组ALP染色结果；B.敲降组ALP染色结果。

Figure 11 ALP staining results in knockdown and control groups（×40）

图12 敲降组与对照组ARS染色结果 A、B.对照组、敲降组ARS染色结果（×40）；C.对照组和敲降组ARS染色OD值比较，**表示P<0.01，与对照组比较。

Figure 12 ARS staining results in knockdown and control groups

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