摘要
目的开发了基于双萤光素酶报告基因稳转细胞株(C2C12~(BBDE))的BMP-2生物学活性新型检测系统,以解决传统碱性磷酸酶(alkaline phosphatase,ALP)活性检测法灵敏度低、耗时长和实时荧光定量聚合酶链反应(qRT-PCR)法操作流程繁琐的问题。方法C2C12^(BBD)_F细胞株通过慢病毒感染,在C2C12细胞中稳定整合了BMP-2响应启动子驱动的纳米萤光素酶(NLuc),并以萤火虫萤光素酶(Fluc)作为内参来确保基础转录水平的稳定性,通过反向转录单元设计和PolyA插入有效消除启动子干扰。结果实验结果表明,新型检测系统具有显著优势:检测灵敏度达0~30 ng/mL,与qRT-PCR相当且显著优于ALP法;检测周期仅需16 h,较ALP法效率提升7倍以上;操作流程简便,避免了qRT-PCR中RNA提取、反转录等复杂步骤。结论该系统的成功开发不仅为BMP-2活性检测提供了标准化工具,其模块化设计更为其他生长因子的活性检测研究提供了可借鉴的技术方案,对促进骨再生药物的研发和临床应用具有重要价值。
Objective To develope a novel BMP-2 bioactivity detection system based on a dual-luciferase reporter gene stably transfected cell line(C2C12^(BBDE))to address the issues of low sensitivity,time-consuming procedures in traditional alkaline phosphatase(ALP)activity assays,and cumbersome operational workflows in real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)methods.Methods This system integrates a BMP-2 responsive promoter driving NanoLuc luciferase(NLuc)expression with constitutively expressed Firefly luciferase(Fluc)as an internal control via lentiviral transduction.Potential promoter interference was mitigated through strategic design of an inverse transcription unit and PolyA sequence insertion.Results This novel system achieved a dynamic detection range of 0-30 ng/mL,matching the sensitivity of qRT-PCR while demonstrating a statistically significant improvement over the ALP assay.Furthermore,the assay cycle was markedly reduced to 16 hours,representing an efficiency enhancement exceeding 700%relative to the ALP method.Critically,the workflow is streamlined,obviating the need for complex RNA extraction and reverse transcription steps inherent in qRT-PCR.Conclusion The successful establishment of this system not only furnishes a standardized,high-throughput tool for BMP-2 bioactivity assessment but also provides a modular,adaptable technical paradigm for the development of bioactivity assays for other growth factors.This advancement holds substantial translational potential,significantly contributing to the rational development and clinical implementation of bone regenerative therapeutics.
作者
符岳阳
夏芷毅
杨建勋
王碧茹
郑瑞
阎博
FU Yueyang;XIA Zhiyi;YANG Jianxun;WANG Biru;ZHENG Rui;YAN Bo(College of Basic Medicine,Fourth Military Medical University,Xi’an,710032,China;Department of Biochemistry and Molecular Biology,Fourth Military Medical University,Xi’an,710032,China;College of Life Science,Yan’an University,Yan’an,Shaanxi,716000,China;Department of Immunology,Fourth Military Medical University,Xi’an,710032,China)
出处
《医学分子生物学杂志》
2025年第5期415-422,共8页
Journal of Medical Molecular Biology
基金
国家肿瘤生物学重点实验室重点课题(No.CBSKL2022ZZ04)
国家肿瘤生物学重点实验室自主课题(No.2025GTEP012)
陕西省重点项目-实验室重点项目(No.2025SYS-SYSZD-030)。
