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背景与目的:APC基因编码的蛋白参与信号转导途径,大量研究证实APC启动子区的高甲基化在肿瘤的发生发展中起重要作用。本研究建立血浆APC基因实时荧光定量甲基化特异性基因扩增(methylation-specificPCR,MSP)检测方法,并对临床肺癌患者血浆进行检测,以确定该方法在肺癌诊断中的应用价值。方法:将APC基因启动子甲基化阳性肺癌细胞株NCI-H460细胞用有限稀释法获取单个集落形成的细胞,以经典的酚-氯仿法提取细胞DNA,并用MSP对APC基因甲基化进行验证,紫外分光光度计定量并以10倍稀释的浓度梯度依次投入到200μL健康人血浆中,得到模拟肺癌患者血浆,利用磁珠核酸提取方法从模拟肺癌及肺癌患者、肺部良性疾病患者和健康对照者血浆中提取DNA,对血浆DNA模板进行亚硫酸氢盐化学修饰;以模拟血浆样品作为标准品,采用外标准曲线法对各种血浆样品中APC甲基化进行定量分析。结果:所建立的实时荧光定量MSP检测方法的线性范围为1.5×102～1.5×105拷贝/mL,用该方法检测甲基化肿瘤细胞的DNA其最低检测限为1.5×102拷贝/mL。78例肺癌患者有40例组织中检测出APC基因甲基化阳性,其中的19例(47.5%)肺癌患者血浆中APC基因启动子甲基化阳性,APC甲基化浓度为1.67×102～6.78×103拷贝/mL,中位浓度为1.60×103拷贝/mL。38例组织阴性的肺癌患者、31例肺部良性疾病患者和23例健康者的血浆APC基因启动子甲基化均为阴性。结论:实时荧光定量MSP检测血浆APC基因甲基化在肺癌诊断方面具有潜在应用价值。
BACKGROUND & OBJECTIVE: The proteins encoded by APC gene are involved in signal transduction pathways. A large number of studies have confirmed that hypermethylation of APC promoter region plays an important role in tumorigenesis and progression. In this study, we established a real-time fluorescence quantitative methylation-specific PCR (MSP) method to detect plasma APC gene in plasma and detected the plasma of clinical lung cancer patients to determine the value of this method in the diagnosis of lung cancer. Methods: Single colony-forming cells were obtained from NCI-H460 cells with APC gene promoter methylation-positive lung cancer cell line. Cell DNA was extracted by classical phenol-chloroform method and the methylation of APC gene was verified by MSP , UV spectrophotometer quantified and 10 times diluted concentration gradient in turn into 200μL of healthy plasma, to obtain simulated plasma of lung cancer patients, the use of magnetic beads nucleic acid extraction method from simulated lung cancer and lung cancer patients with benign lung disease and healthy controls Plasma DNA was extracted and the DNA templates were chemically modified with bisulfite. The plasma samples were used as standard samples and the APC methylation in various plasma samples was quantitatively analyzed by external standard curve method. Results: The linear range of real-time fluorescence quantitative MSP assay was 1.5 × 102 ~ 1.5 × 105 copies / mL. The minimum detectable limit of detection of methylated tumor cells by this method was 1.5 × 102 copies / mL. APC gene methylation was detected in 40 out of 78 cases of lung cancer, of which 19 (47.5%) had positive APC gene promoter methylation in plasma of APC patients, the APC methylation concentration was 1.67 × 102 ~ 6.78 × 103 copies / mL at a median concentration of 1.60 × 103 copies / mL. Thirty-eight patients with negative tissue lung cancer, 31 patients with benign pulmonary disease, and 23 healthy controls were negative for plasma APC gene promoter methylation. Conclusion: Real-time fluorescence quantitative MSP detection of plasma APC gene methylation has potential value in the diagnosis of lung cancer.