中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (31): 8100-8107.doi: 10.12307/2026.848

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

机械模量调控鼻黏膜外胚层间充质干细胞的衰老

周金龙1，2，王  娟2，卞  璐2，3，吕  龙2，阙云端1，钱文武1   

  1. 1江苏卫生健康职业学院附属南京市高淳人民医院骨科，江苏省南京市  211300；2江苏大学附属高淳医院中心实验室，江苏省南京市  211300；3江南大学无锡医学院，江苏省无锡市  214026
  • 收稿日期:2025-09-26 接受日期:2026-02-13 出版日期:2026-11-08 发布日期:2026-05-23
  • 通讯作者: 钱文武，硕士生导师，副主任医师，江苏卫生健康职业学院附属南京市高淳人民医院骨科，江苏省南京市 211300
  • 作者简介:周金龙，男，1989年生，江苏省南京市人，汉族，南京医科大学在读硕士，主治医师，主要从事骨外科研究。
  • 基金资助:
    江苏卫生健康职业学院校级科研项目(YJXTG202309)，项目负责人：周金龙；南京市卫生科技发展专项资金项目(重点项目：ZKX21063)，项目负责人：阙云端

Modulation of nasal mucosa-derived ectomesenchymal stem cell senescence by mechanical modulus

Zhou Jinlong1, 2, Wang Juan2, Bian Lu2, 3, Lyu Long2, Que Yunduan1, Qian Wenwu1   

  1. 1Department of Orthopedics, Nanjing Gaochun People’s Hospital Affiliated to Jiangsu Health Vocational College, Nanjing 211300, Jiangsu Province, China; 2Central Laboratory, Gaochun Hospital Affiliated to Jiangsu University, Nanjing 211300, Jiangsu Province, China; 3Wuxi Medical School, Jiangnan University, Wuxi 214026, Jiangsu Province, China
  • Received:2025-09-26 Accepted:2026-02-13 Online:2026-11-08 Published:2026-05-23
  • Contact: Qian Wenwu, Master’s supervisor, Associate chief physician, Department of Orthopedics, Nanjing Gaochun People’s Hospital Affiliated to Jiangsu Health Vocational College, Nanjing 211300, Jiangsu Province, China
  • About author:Zhou Jinlong, MS candidate, Attending physician, Department of Orthopedics, Nanjing Gaochun People’s Hospital Affiliated to Jiangsu Health Vocational College, Nanjing 211300, Jiangsu Province, China; Central Laboratory, Gaochun Hospital Affiliated to Jiangsu University, Nanjing 211300, Jiangsu Province, China
  • Supported by:
    Jiangsu Health Vocational College Institutional Research Project, No. YJXTG202309 (to ZJL); Nanjing Municipal Health Science and Technology Development Special Fund Project (Key Project), No. ZKX21063 (to QYD)

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摘要：

文题释义：

机械模量：医学中主要表征生物组织或者细胞载体支架的软硬程度，是评估生物力学性能的关键参数，其中较为常用的是储存模量、损失模量和弹性模量。细胞可通过膜表面受体感知周围基质的机械模量，并将机械信号转化为化学或者生物信号，从而直接影响细胞增殖、分化和迁移。精准调控支架材料的机械模量，对诱导干细胞定向分化及实现功能性再生至关重要。
鼻黏膜外胚层间充质干细胞：是分布于头面部黏膜内的特殊类型的干细胞，来源于胚胎时期的神经嵴，该细胞仍保留多向分化潜能。体外传代和扩增的鼻黏膜外胚层间充质干细胞仍高表达干细胞标志蛋白，并维持细胞干性。鼻黏膜外胚层间充质干细胞与其他成体干细胞一致，具有促进组织损伤修复的功能。

摘要
背景：细胞微环境的力学特性在调控鼻黏膜外胚层间充质干细胞命运中发挥着关键作用。
目的：探讨机械模量对鼻黏膜外胚层间充质干细胞衰老过程的调控效应及潜在的分子机制。
方法：配制50，100，150 g/L 3种质量浓度的甲基丙烯酸化明胶水凝胶，设置为低、中、高机械模量组，以普通培养板作为对照组。分离培养并鉴定SD大鼠鼻黏膜外胚层间充质干细胞，以第5代细胞为起点种植于不同机械模量的水凝胶上，持续培养至第7代，并分别给予Piezo1激活剂(Yoda1)和谷氨酰转氨酶2抑制剂(KCC009)干预。采用RT-qPCR、免疫印迹和免疫荧光检测以下指标：衰老相关基因和蛋白(p16和p21)表达；增殖相关基因(Mki67、Pcna)表达；成骨分化标志物(Spp1、Sp7和Ⅰ型胶原蛋白)表达；Piezo1、钙离子及谷氨酰胺转氨2表达；纤维化相关蛋白(α-平滑肌肌动蛋白和Ⅰ型胶原蛋白)表达。采用β-半乳糖苷酶染色检测衰老细胞比例，碱性磷酸酶染色检测碱性磷酸酶活性，茜素红S染色检测钙结节形成情况。
结果与结论：低机械模量水凝胶可显著下调衰老相关标志物p21和p16的表达，降低β-半乳糖苷酶阳性细胞比例，同时抑制细胞纤维化，增强成骨分化能力；相比之下，高机械模量水凝胶则呈现相反趋势。进一步研究发现，种植于高机械模量水凝胶上的鼻黏膜外胚层间充质干细胞中Piezo1表达上调，钙离子内流增强，谷氨酰胺转胺酶2表达升高。抑制谷氨酰胺转胺酶2表达可有效延缓高机械模量水凝胶所诱导的外胚层间充质干细胞衰老进程。结果表明：低机械模量微环境可逆转鼻黏膜外胚层间充质干细胞衰老，Piezo1/谷氨酰胺转氨2信号通路在此过程中发挥关键调控作用。

关键词: 鼻黏膜外胚层间充质干细胞, 机械模量, 衰老, piezo1, 谷氨酰胺转胺酶2, 甲基丙烯酸化明胶, 成骨分化, 纤维化

Abstract: BACKGROUND: The mechanical properties of the cellular microenvironment play a pivotal role in regulating the fate of nasal mucosa–derived ectomesenchymal stem cells.
OBJECTIVE: To investigate the regulatory effects of mechanical modulus on the senescence of ectomesenchymal stem cells and the potential molecular mechanisms.
METHODS: Gelatin methacryloyl hydrogels with 50, 100, and 150 g/L mass concentrations were fabricated, and low-, medium-, and high-mechanical moduli were established, with standard culture plates serving as controls. Ectomesenchymal stem cells of SD rats were isolated, and identified. Passage 5 cells were seeded onto hydrogels of different moduli and cultured to passage 7. Cells were further treated with the Piezo1 activator Yoda1 or the transglutaminase 2 inhibitor KCC009. RT-qPCR, western blotting, and immunofluorescence were performed to assess the expression of senescence markers (p16 and p21), proliferation-associated genes (Mki67 and Pcna), osteogenic markers (Spp1, Sp7, type I collagen), Piezo1, calcium ions, transglutaminase 2, and fibrotic proteins (α-smooth muscle actin and type I collagen). β-Galactosidase staining was used to quantify the proportion of senescent cells. Alkaline phosphatase staining was used to measure alkaline phosphatase activity. Alizarin Red S staining was applied to assess mineralized nodule formation.
RESULTS AND CONCLUSION: Low-modulus hydrogels significantly downregulated p16 and p21 expression, reduced β-galactosidase–positive cells, alleviated fibrosis, and enhanced osteogenic differentiation, whereas high-modulus hydrogels induced the opposite effects. Further mechanistic analysis revealed that ectomesenchymal stem cells cultured on high-modulus substrates exhibited increased Piezo1 expression, enhanced Ca²⁺ influx, and elevated transglutaminase 2 levels. Inhibition of transglutaminase 2 effectively attenuated high-modulus–induced ectomesenchymal stem cell senescence. These findings indicate that a low-modulus microenvironment can reverse ectomesenchymal stem cell senescence, and the Piezo1/transglutaminase 2 signaling pathway serves as a key regulator in this process.

Key words: ">nasal mucosa–derived ectomesenchymal stem cells, mechanical modulus, senescence, piezo1, transglutaminase 2, gelatin methacryloyl, osteogenic differentiation, fibrosis

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