摘要
目的开发一种基于滚环扩增(rolling circle amplification,RCA)技术的高灵敏度外周血G-四链体(guanine quadruplex,G4)检测方法,建立ATRX突变型脑胶质瘤的无创分子诊断体系,为临床早期筛查提供新型液体活检策略。方法采用shRNA慢病毒感染技术建立ATRX稳定敲低的原代脑胶质瘤细胞株(ATRX KD),通过硫黄素T(thioflavin T,ThT)荧光光谱法检测细胞上清液中G4的形成水平。构建ATRX敲低的原位脑胶质瘤小鼠模型,采用RCA变温扩增技术检测模型组与对照组外周血循环游离DNA(cfDNA)中G4的丰度,并对RCA变温扩增技术的程序进行优化。收集临床确诊的ATRX突变型和野生型胶质瘤患者,以及健康志愿者的血清样本,提取cfDNA后采用优化的RCA方案进行G4定量检测,并再次对检测程序进行优化。结果体外实验显示,ATRX敲低可显著促进G4结构的形成,ATRX KD组细胞上清液中ThT荧光强度较野生型显著增加。建立RCA变温扩增方案检测ATRX突变型胶质瘤小鼠模型外周血cfDNA中G4结构的实验条件:以50 ng cfDNA为起始模板、进行10个循环扩增。临床样本验证显示,优化后的RCA变温扩增方案较传统恒温扩增效率提升,能够有效区分ATRX突变型与野生型胶质瘤患者及健康人群。结论成功建立了基于RCA变温扩增的外周血G4高灵敏度检测方案,该技术首次实现ATRX突变状态的液体活检分子分型,为胶质瘤早期筛查提供具有临床应用前景的无创诊断方法。
Objective To develop a highly sensitive method for detecting peripheral blood G」quadruplex(G4)based on rolling circle amplification(RCA)technology,establish a non-invasive molecular diagnostic system for ATRX-mutant gliomas,and provide a novel liquid biopsy strategy for clinical early screening.Methods ATRX knockdown glioma cell lines(ATRX KD)were constructed via shRNA lentiviral infection.Thioflavin T(ThT)fluorescence spectroscopy was employed to detect G4levels in the supernatant.An orthotopic ATRX knockdown glioma mouse model was established.RCA was applied to compare G4 abundance in peripheral blood circulating cell-free DNA(cfDNA)between model and control groups,with optimization of RCA amplification protocols.Serum samples from clinically confirmed ATRX-mutant gliomas,wild-type gliomas,and healthy volunteers were collected.After cfDNA extraction,G4 quantification was performed using optimized RCA protocols with further procedural refinement.Results In vitro experiments demonstrated that ATRX knockdown significantly promoted the formation of G4 structures,with the ThT fluorescence intensity in the supernatant of A TRX KD cells being significantly higher than that in wild-type cells.The experimental conditions for detecting G4 structures in cfDNA from the peripheral blood of ATRX-mutant glioma mouse models using RCA temperature-variable amplification were established:starting with 50 ng of cfDNA as the template and performing 10 amplification cycles.Clinical sample validation showed that the optimized RCA temperaturevariable amplification protocol improved efficiency compared to traditional isothermal amplification and effectively distinguished ATRX-mutant glioma patients from wild-type patients and healthy individuals.Conclusion This study successfully developed a highly sensitive peripheral blood G4 detection system based on RCA.This technology pioneers liquid biopsy-based molecular subtyping of ATRX mutation status,offering a clinically promising non-invasive diagnostic approach for early glioma screening.
作者
杨华
陈姝雨
刘秀萍
王宇翔
YANG Hua;CHEN Shu-yu;LIU Xiu-ping;WANG Yu-xiang(Department of Pathology,School of Basic Medical Sciences,Fudan University,Shanghai 200032,China;Department of Pathology,Shanghai Fifth People's Hospital,Shanghai 200240,China)
出处
《复旦学报(医学版)》
北大核心
2026年第2期202-210,216,共10页
Fudan University Journal of Medical Sciences
