中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (13): 3298-3307.doi: 10.12307/2026.728

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

低氧状态下miR-223-3p对肌腱干细胞生物学行为的影响

段  成，程  杰   

  1. 内蒙古医科大学第二附属医院创伤外科中心C区，内蒙古自治区呼和浩特市  010030
  • 接受日期:2025-09-18 出版日期:2026-05-08 发布日期:2025-12-25
  • 通讯作者: 程杰，硕士，主任医师，内蒙古医科大学第二附属医院创伤外科中心C区，内蒙古自治区呼和浩特市 010030
  • 作者简介:段成，男，1999年生，内蒙古自治区呼和浩特市人，汉族，内蒙古医科大学在读硕士，主要从事四肢创伤外科研究。 共同第一作者：程杰，男，1983年生，山东省荷泽市人，汉族，硕士，主任医师，主要从事四肢创伤外科研究。
  • 基金资助:
    内蒙古自治区首府地区公立医院高水平临床专科建设科技项目(2024SGGZ129)，项目负责人：程杰；内蒙古医科大学面上项目(YKD2024MS021)，项目负责人：程杰

Effect of miR-223-3p on biological behavior of tendon stem cells under hypoxic conditions

Duan Cheng, Cheng Jie   

  1. Department of Trauma Area C, Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
  • Accepted:2025-09-18 Online:2026-05-08 Published:2025-12-25
  • Contact: Cheng Jie, MS, Chief physician, Department of Trauma Area C, Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
  • About author:Duan Cheng, Master candidate, Department of Trauma Area C, Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China; Cheng Jie, MS, Chief physician, Department of Trauma Area C, Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China Duan Cheng and Cheng Jie contributed equally to this article.
  • Supported by:
    Science and Technology Project for the Construction of High-level Clinical Specialties in Public Hospitals in the Capital Area of Inner Mongolia Autonomous Region, No. 2024SGGZ129 (to CJ); General Project of Inner Mongolia Medical University, No. YKD2024MS021 (to CJ)

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摘要：

文题释义：

miRNA：在真核生物中由19-25个核苷酸组成的非编码小分子 RNA，主要参与细胞的增殖、分化和凋亡等生理活动。初级转录物经过一系列核酸酶的剪切加工产生成熟的miRNA，通过碱基互补配对的方式识别靶基因mRNA，沉默复合体降解靶基因或阻遏靶基因的翻译。
肌腱干细胞：是从肌腱组织中分离出的具有自我更新和多向分化潜能的成体干细胞，是促进肌腱愈合的潜在种子细胞。

摘要
背景：低氧微环境是肌腱损伤修复的关键调控因素，但其通过miRNA介导的分子机制尚不明确。
目的：探讨miR-223-3p在低氧条件下对肌腱干细胞生物学行为的调控作用及机制。
方法：①将第3代大鼠肌腱干细胞分为对照组(体积分数21%氧气)、低氧组(体积分数1%氧气)，培养48 h采用CCK-8实验检测细胞增殖情况，Muse细胞凋亡检测试剂盒检测细胞凋亡率，RT-qPCR及Western blot检测低氧诱导因子1α、血管内皮生长因子蛋白及mRNA表达。②生物信息学预测miR-223-3p靶点，双荧光素酶实验验证miR-223-3p与VHL的靶向调控关系。③将第3代大鼠肌腱干细胞分为5组：常氧miR-223-3p mimics组、常氧mimics NC组、常氧inhibitor NC组、低氧inhibitor NC组、低氧miR-223-3p inhibitor组，常氧或低氧培养48 h采用RT-qPCR及Western blot检测低氧诱导因子1α、血管内皮生长因子、VHL蛋白及mRNA表达，CCK-8法检测细胞活力，Muse细胞凋亡检测试剂盒分析细胞凋亡率，划痕实验及Transwell小室实验评估细胞迁移与侵袭能力。
结果与结论：①与对照组相比，低氧组肌腱干细胞活力显著降低(P < 0.001)，凋亡率显著增加(P < 0.01)；与对照组相比，低氧组肌腱干细胞中miR-223-3p、低氧诱导因子1α、血管内皮生长因子mRNA表达显著升高(P < 0.01)；低氧组肌腱干细胞中低氧诱导因子1α、血管内皮生长因子蛋白表达显著升高(P < 0.01)；②生物信息学预测VHL与miR-223-3p有潜在结合位点；双荧光素酶实验显示，miR-223-3p能够与VHL靶向结合；③功能实验显示，敲低miR-223-3p可上调肌腱干细胞中VHL mRNA和蛋白表达，下调低氧诱导因子1α、血管内皮生长因子mRNA和蛋白表达(P < 0.01)，提高肌腱干细胞活力，降低细胞凋亡率，促进细胞迁移及侵袭(P < 0.01)，过表达miR-223-3p则相反，且加剧低氧损伤，其机制可能与低氧诱导因子1α/血管内皮生长因子/VHL信号通路被激活相关。

关键词: 肌腱干细胞, miR-223-3p, 低氧诱导因子1α, 血管内皮生长因子, VHL, 低氧环境

Abstract: BACKGROUND: The hypoxic microenvironment is a key regulator of tendon injury repair, but the molecular mechanism mediated by microRNAs (miRNAs) is not well understood.
OBJECTIVE: To investigate the regulatory role and mechanism of miR-223-3p on the biological behavior of tendon stem cells under hypoxic conditions. 
METHODS: (1) The rat third-generation tendon stem cells were divided into control group (O₂ concentration 21%) and hypoxia group (O₂ concentration 1%). After 48 hours of culture, CCK-8 assay was used to detect cell proliferation. Muse apoptosis detection kit was used to detect apoptosis rate. RT-qPCR and western blot assay were used to detect the protein and mRNA expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor. (2) miR-223-3p targets were predicted by bioinformatics. The targeting and regulation relationship between miR-223-3p and VHL was verified by double luciferase assay. (3) The third-generation rat tendon stem cells were divided into five groups: normoxic miR-223-3p mimic group, normoxic mimic negative control group, normoxic inhibitor negative control group, hypoxic inhibitor negative control group, and hypoxic miR-223-3p inhibitor group. After culture under normoxia or hypoxia for 48 hours, the expression of hypoxia-inducible factor-1α, vascular endothelial growth factor, and VHL protein and mRNA was detected by RT-qPCR and western blot assay. Cell viability was determined by CCK-8 assay, and the apoptosis rate was analyzed by Muse cell apoptosis detection kit. The cell migration and invasion were evaluated by scratch assay and Transwell chamber assay.
RESULTS AND CONCLUSION: (1) Compared with the control group, the tendon stem cell viability in the hypoxic group was significantly decreased (P < 0.001), and the apoptosis rate was significantly increased (P < 0.01). Compared with the control group, the mRNA expressions of miR-223-3p, hypoxia-inducible factor-1α and vascular endothelial growth factor in tendon stem cells in the hypoxic group were significantly increased (P < 0.01). The expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor proteins in tendon stem cells in the hypoxia group were significantly increased (P < 0.01). (2) Bioinformatics predicted that VHL had potential binding sites with miR-223-3p. Dual-luciferase assay showed that miR-223-3p could bind to VHL targeting. (3) Functional experiments showed that knockdown of miR-223-3p could up-regulate the mRNA and protein expression of VHL in tendon stem cells, down-regulate the mRNA and protein expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor (P < 0.01), improve the viability of tendon stem cells, reduce the rate of apoptosis, and promote cell migration and invasion (P < 0.01), while overexpression of miR-223-3p was the opposite and aggravated hypoxic damage, which may be related to the activation of hypoxia-inducible factor-1α/vascular endothelial growth factor/VHL signaling pathway. 

Key words: ">tendon stem cell, miR-223-3p, hypoxia-inducible factor-1α, vascular endothelial growth factor, VHL, hypoxic environment

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