Yoda1对人施耐德膜来源间充质细胞成骨分化影响的实验研究

doi:10.12439/kqhm.1005-4979.2026.03.002

摘要/Abstract

摘要：

目的：

探讨PIEZO1特异性激动剂Yoda1对人施耐德膜（human Schneider membrane，hSM）来源间充质细胞成骨分化的影响。

方法：

采用酶消化法分离培养hSM来源细胞，通过流式细胞术鉴定细胞表面标志物，免疫荧光染色检测角蛋白14（keratin14，KRT14）和组织蛋白酶K（cathepsin K，CTSK）的表达，蛋白质印迹法检测细胞PIEZO1蛋白表达。利用钙离子荧光探针检测Yoda1对PIEZO1通道的激活作用。通过CCK-8法和划痕实验评估不同浓度Yoda1对hSM来源间充质细胞增殖和迁移能力的影响，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）和蛋白质印迹法检测碱性磷酸酶（alkaline phosphatase，ALP）基因ALPL和Runt相关转录因子2（Runt-related transcription factor 2，RUNX2）的表达，结合ALP染色和茜素红S染色评价细胞成骨分化能力。

结果：

hSM来源细胞表达间充质细胞表面标志物，共表达KRT14和CTSK蛋白。细胞中检测到PIEZO1蛋白表达，Yoda1可有效激活PIEZO1通道。低浓度Yoda1对细胞增殖无显著影响，而高浓度Yoda1（10.0 μmol/L）可显著抑制细胞增殖（P<0.05），但对细胞迁移无显著影响。成骨诱导过程中，低浓度Yoda1（0.1 μmol/L）可上调ALPL基因表达并增强ALP活性，而对RUNX2表达影响不明显；蛋白质印迹法结果与mRNA表达趋势一致。ALP染色及茜素红染色结果显示，低浓度Yoda1对成骨分化具有一定促进作用。

结论：

Yoda1对hSM来源间充质细胞成骨分化作用有限，低浓度Yoda1可能促进其早期成骨分化。

关键词: Yoda1, PIEZO1, 施耐德膜, 成骨分化, 间充质细胞, 上颌窦底提升术

Abstract:

Objective:

To investigate the effect of the PIEZO1-specific agonist Yoda1 on osteogenic differentiation of mesenchymal cells derived from human Schneiderian membrane (hSM).

Methods:

hSM-derived cells were isolated and cultured by enzymatic digestion. Cell surface markers were identified by flow cytometry, and the expression of keratin 14 (KRT14) and cathepsin K (CTSK) was detected by immunofluorescence staining. PIEZO1 protein expression was evaluated by Western blotting, and a calciumion fluorescent probe was used to detect activation of the PIEZO1 channels by Yoda1. The effects of different concentrations of Yoda1 on the proliferation and migration abilities of hSM-derived mesenchymal cells were evaluated using CCK-8 and wound healing assays, respectively. Following osteogenic induction, the expression levels of alkaline phosphatase (ALP) gene ALPL and Runt-related transcription factor 2 (RUNX2) were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ALP staining and alizarin red S staining were performed to evaluate the osteogenic differentiation ability of the cells.

Results:

hSM-derived cells expressed mesenchymal cell surface markers and co-expressed KRT14 and CTSK proteins. PIEZO1 protein was detected in these cells, and Yoda1 effectively activated the PIEZO1 channels. Low concentrations of Yoda1 had no significant effect on cell proliferation, whereas a high concentration (10.0 μmol/L) significantly inhibited proliferation (P<0.05). No significant effect on cell migration was observed. During osteogenic induction, low-concentration Yoda1 (0.1 μmol/L) upregulated ALPL expression and enhanced ALP activity, while no significant change was observed in RUNX2 expression. Western blotting results were consistent with mRNA expression trends. ALP staining and alizarin red S staining indicated that low-concentration Yoda1 exerted a modest promoting effect on osteogenic differentiation.

Conclusion:

Yoda1 exhibits a limited promoting effect on osteogenic differentiation of hSM-derived mesenchymal cells, and low concentrations may promote its early osteogenic differentiation.

Key words: Yoda1, PIEZO1, Schneiderian membrane, osteogenic differentiation, mesenchymal cells, maxillary sinus floor elevation

李泽远, 王佐林. Yoda1对人施耐德膜来源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(3): 177-185.

LI Zeyuan, WANG Zuolin. Effect of Yoda1 on osteogenic differentiation of human Schneiderianmembrane-derived mesenchymal cells[J]. Journal of Oral and Maxillofacial Surgery(CN), 2026, 36(3): 177-185.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2026/V36/I3/177

图/表 5

表1 RT-qPCR引物序列

Table 1 Primer sequences used in RT-qPCR

基因	正向引物(5'-3')	反向引物(5'-3')
β-actin	CATGTACGTTGCTATCCAGGC	CTCCTTAATGTCACGCACGAT
PIEZO1	ATGTTGCTCTACACCCTGACC	CCAGCACACACATAGATCCAGT
ALPL	ACCACCACGAGAGTGAACCA	CGTTGTCTGAGTACCAGTCCC
RUNX2	TGGTTACTGTCATGGCGGGTA	TCTCAGATCGTTGAACCTTGCTA

图1 hSM来源间充质细胞的培养鉴定及PIEZO1通道的表达与激活 A. P3代hSM来源细胞形态（×40）；B、C.流式细胞术检测细胞表面标志物表达；D. KRT14和CTSK免疫荧光染色图（×40）；E. PIEZO1免疫荧光染色图（×100）；F.蛋白质印迹法检测PIEZO1蛋白表达（Hela细胞为阳性对照）；G.钙离子荧光探针Fluo-4/AM检测Yoda1处理前后细胞内Ca²⁺水平（×40）。

Figure 1 Culture and identification of hSM-derived mesenchymal cells and expression and activation of the PIEZO1 channel

图2 不同浓度Yoda1对hSM来源间充质细胞增殖和迁移能力的影响 A.CCK-8法检测结果示不同浓度Yoda1处理后的细胞增殖曲线；B.不同浓度Yoda1培养第7天时的细胞存活率；C.划痕实验示0、24 h的细胞迁移情况（×40）；D.划痕愈合率的定量分析。*表示P<0.05，与0 μmol/L组比较。

Figure 2 Effects of different concentrations of Yoda1 on the proliferation and migration abilities of hSM-derived mesenchymal cells

图3 成骨诱导不同时间点hSM来源间充质细胞PIEZO1、ALPL、RUNX2基因表达变化 A.成骨诱导7 d后2组细胞PIEZO1、ALPL、RUNX2 mRNA表达水平；B.成骨诱导14 d后2组细胞PIEZO1、ALPL、RUNX2 mRNA表达水平。*表示P<0.05，***表示P<0.001，与对照组比较。

Figure 3 Changes in the expression of PIEZO1, ALPL, and RUNX2 genes in hSM-derived mesenchymal cells at different time points after osteogenic induction

图4 不同浓度Yoda1对hSM来源间充质细胞成骨分化的影响 A、B.分别为成骨诱导7、14 d后，各组ALPL、RUNX2 mRNA表达水平；C.成骨诱导14 d后，各组RUNX2蛋白表达水平；D.成骨诱导14 d后，各组ALP染色结果；E.成骨诱导28 d后，各组茜素红S染色结果。*表示P<0.05，**表示P<0.01，***表示P<0.001，与0 μmol/L组比较。

Figure 4 Effects of different concentrations of Yoda1 on osteogenic differentiation of hSM-derived mesenchymal cells

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