中国药物警戒 ›› 2026, Vol. 23 ›› Issue (2): 121-126.
DOI: 10.19803/j.1672-8629.20250916

• 基础与临床研究 • 上一篇    下一篇

端粒酶活性检测RQ-TRAP方法学验证及在间充质干细胞检测评价中的应用研究

张真1,2, 喻谢安1,2, 许淳淇1,2, 陈宁1,2, 秦美蓉1,2,*, 王平1,2#   

  1. 1深圳市药品检验研究院药理毒理室，广东 深圳 518000;
    2广东省细胞与基因治疗创新药物质控工程技术研究中心，广东 深圳 518000
  • 收稿日期:2025-12-19 出版日期:2026-02-15 发布日期:2026-02-13
  • 通讯作者: *秦美蓉，女，硕士，主任药师，传统药品与先进治疗药品检测评价。E-mail: szqinmeirong@sina.com; #为共同通信作者。
  • 作者简介:张真，男，博士，副主任药师，先进治疗药品检测评价。
  • 基金资助:
    国家自然科学基金资助项目（82104357）

Methodological Validation of RQ-TRAP for Telomerase Activity Detection and Its Applications in Human Mesenchymal Stem Cells

ZHANG Zhen1,2, YU Xie'an1,2, XU Chunqi1,2, CHEN Ning1,2, QIN Meirong1,2,*, WANG Ping1,2#   

  1. 1Department of Pharmacology and Toxicology, Shenzhen Institute for Drug Control, Shenzhen Guangdong 518000, China;
    2Guangdong Engineering Technology Research Center for Quality Control of Cell and Gene Therapy Products, Shenzhen Guangdong 518000, China
  • Received:2025-12-19 Online:2026-02-15 Published:2026-02-13

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摘要： 目的 验证实时定量端粒酶重复扩增方法（Real-Time Quantitative Telomerase Repeat Amplification Protocol, RQ-TRAP）的方法学性能，包括专属性、准确性、精密度、线性、扩增效率、定量下限（Lower Limit of Quantification, LLOQ），并将其应用于人源间充质干细胞（Human Mesenchymal Stem Cells, hMSCs）的端粒酶活性检测评价，以评估hMSCs的增殖潜能、衰老进程及潜在肿瘤转化风险，为再生医学中的细胞疗法提供标准化检测手段。方法 采用RQ-TRAP方法对hMSCs进行端粒酶活性检测。hMSCs来源于人脐带，培养于MEM-α培养液中。样品制备包括细胞裂解提取，RQ-TRAP体系使用TRAP Reaction Buffer等试剂，荧光定量聚合酶链式反应仪进行绝对定量检测。方法学验证使用TSR8标准品进行专属性、准确性（回收率）、中间精密度（RSD）、线性（R²和扩增效率）和LLOQ（回收率置信区间）评估。hMSCs检测包括标准曲线拟合和Ct值计算端粒酶活性。结果 方法学验证显示专属性良好，无热灭活细胞基质干扰;准确性回收率95%置信区间为91.37%~111.0%（符合75%~125%）;中间精密度RSD均<25%;线性R2>0.99，扩增效率104.1%（90%~110%）;LLOQ为0.2 TPG Units（回收率置信区间70%~130%）。hMSCs端粒酶活性为1.432 TPG/万个细胞，标准曲线扩增效率102.6%。结论 RQ-TRAP方法经验证可靠、灵敏，适用于hMSCs端粒酶活性定量检测，可有效评估细胞衰老和肿瘤风险。该方法填补hMSCs标准化检测空白，支持再生医学安全应用。

关键词: 端粒酶活性, 间充质干细胞, 成瘤性检测, 荧光定量PCR, 方法学验证

Abstract: Objective To validate the methodological performance of the real-time quantitative telomeric repeat amplification protocol (RQ-TRAP), including the specificity, accuracy, precision, linearity, amplification efficiency, and lower limit of quantification, and detect telomerase activity in human mesenchymal stem cells (hMSCs) using this method in order to assess the proliferative potential, senescence process, and potential tumorigenic risk of hMSCs. Methods Telomerase activity in hMSCs was detected using the RQ-TRAP method. hMSCs derived from human umbilical cords were cultured in MEM-α medium. Samples were prepared via cell lysis and extraction, and absolute quantification was performed on a real-time polymerase chain reaction system using TRAP reaction buffer. Methodological validation was conducted using TSR8 control templates to evaluate the specificity, accuracy (recovery rate), intermediate precision (RSD), linearity (R2 and amplification efficiency), and LLOQ (confidence interval of recovery). For hMSCs assessment, telomerase activity was calculated based on standard curve fitting and Ct values. Results Methodological validation results indicated a good specificity with no interference from heat-inactivated cell matrices. The 95% confidence interval (CI) for accuracy recovery ranged from 91.37% to 111.0% (meeting the standard of 75%-125%). The intermediate precision (RSD) was less than 25% for all samples. Linearity assessment showed an R2>0.99 with an amplification efficiency of 104.1% (within the 90%-110% range). The LLOQ was determined to be 0.2 TPG Units (recovery CI: 70%-130%). The telomerase activity in hMSCs was measured at 1.432 TPG per 10 000 cells and the amplification efficiency of the standard curve in this assay was 102.6%. Conclusion The RQ-TRAP method has proved to be reliable and sensitive, which can be used for quantitative detection of telomerase activity in hMSCs and for effective assessment of cell senescence and tumorigenic risks. This method fills the gap in standardized testing for hMSCs and supports safe applications of regenerative medicine.

Key words: Telomerase Activity, Mesenchymal Stem Cells, Methodological Validation, Real-time Quantitative PCR, Tumorigenicity Assessment

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