乳腺癌肿瘤标志物血浆cell-free DNA含量的Sybr Green荧光定量分析  

Quantitative analysis of a liquid tumor biomarker circulating cell-free DNA in breast cancers using sybr green-based fluorescent assay

作　　者：王艺斐[1,2] 王学健[1,3] 路中[2] 史立宏[1,3] 王丽华[1,2] 丁怡[1] WANG Yifei;WANG Xuejian;LU Zhong;SHI Lihong;WANG Lihua;DING Yi(Molecular Oncology Laboratory,Weifang Medical University,Weifang 261053,China;the Affiliated Hospital of Weifang Medical University,3Department of Pharmacology;Weifang Medical University)

机构地区：[1]潍坊医学院分子肿瘤学实验室,山东潍坊261053 [2]潍坊医学院附属医院 [3]潍坊医学院药理学教研室

出　　处：《潍坊医学院学报》2019年第4期241-243,F0003,共4页Acta Academiae Medicinae Weifang

基　　金：国家自然科学基金资助课题(项目编号:81472489);山东省科技发展计划项目资助课题(项目编号:2014GGB14403);山东省高等学校科技计划项目资助课题(项目编号:J13LK02);国家留学回国人员科研启动基金资助项目

摘　　要：目的建立一种基于Sybr Green染色的cfDNA荧光定量法定量分析血浆中肿瘤液体标记物cfDNA的含量,并评估在临床乳腺癌患者和动物模型中的应用价值。方法从血浆中提取的cfDNA加入Sybr Green染料读取荧光值对其定量,同时应用qPCR定量检测cfDNA中管家基因GAPDH与之对比,并在从4T1乳腺癌自发转移性小鼠动物模型和临床乳腺癌患者的血液样品中进一步验证。结果 Sybr Green荧光染料法测得的cfDNA含量与由qPCR实验所获得的结果呈一致性。临床样本和动物模型均显示乳腺癌组的血浆cfDNA含量明显高于健康对照组。且在4T1乳腺癌自发转移性小鼠动物模型中发现其血浆cfDNA浓度与乳腺癌瘤重之间呈正线性相关。结论基于Sybr Green染色的cfDNA荧光定量法是一种简单、稳定、可靠和敏感的分析方法,不仅可用于定量分析临床乳腺癌患者和动物模型中血浆肿瘤液体标记物cfDNA的含量,而且cfDNA定量分析还可以作为反映肿瘤负荷的有效指标。Objective To establish a fluorescent dye-based method for the quantification of cfDNA and assessment of the clinical significance in breast cancer.Methods Sybr Green fluorescent dye was added to the extracted cfDNA from plasma.And the fluorescence value was read to quantify the cfDNA.As control,we also quantify the cfDNA using qPCR by detecting the GAPDH sequence.The method was validated in blood samples obtained from both animal model and clinical patients.Results The experiment using the tumor cellular DNA spiking into blood showed that the value obtained from Sybr Green fluorescent dye-based assay was compatible to the data acquired by qPCR assay.By using this method,it was found that the cfDNA concentration in tumor subjects were significantly higher than that in healthy subjects in both clinical patients and animal models.More interestingly,the concentrations of circulating cfDNA was correlated to the tumor weight and size developed in the 4 T1 breast cancer mouse model.Conclusion Sybr Green-based cfDNA quantitation method is robust assay and enable to distinguish tumor patients from healthy people using blood-derived samples.These data provide evidence that the Sybe Green fluorescent dye method is a reliable quantitative assay for detecting cfDNA amount as a liquid tumor biomarker in breast cancer.

关 键 词：乳腺癌 循环游离DNA SYBR Green 定量PCR 

分 类 号：R34[医药卫生—基础医学]