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目的 建立抑癌基因APC启动子部位甲基化的检测方法 ,并对肺癌临床样品进行初步检测研究。方法 :对肺癌组织提取的DNA及转甲基后的脐带血DNA进行化学修饰 ,以甲基化特异性引物进行基因扩增 (methylationspecificPCR ,MSP)和甲基化序列分析 (methylationsequencing ,MS)。结果 :经转甲基的人脐带血DNA样品MSP检测为阳性 ,其序列与预期相符 ,未经转甲基样品为阴性。 17例肺癌患者的肿瘤及其正常肺组织APC甲基化检测阳性率分别为 4 7% (8/ 17)和 18% (3/ 17) ,1例肺部良性肿瘤及其正常肺组织的检测结果均为阴性 ;对MSP检测为阴性的 9例肺癌患者肿瘤及其正常肺组织进行MS检测分析 ,有 4例APC启动子部位存在CpG甲基化。结论 :利用MSP和MS两种甲基化检测分析手段 ,对 17例临床确诊肺癌组织的检测总阳性率可达 71% (12 / 17)。MSP操作简便 ,价格低廉 ,可适合于大规模肿瘤患者样本的筛查 ;而对于MSP检测为阴性的临床样品 ,可以利用MS进行进一步的确认 ,为临床肺癌诊断提供更准确的信息。
OBJECTIVE: To establish a method for detecting methylation of the promoter region of tumor suppressor gene APC, and to conduct preliminary detection of clinical samples of lung cancer. Methods: DNA extracted from lung cancer tissues and cord blood DNA after methyltransferase were chemically modified, and methylationspecific PCR (MSP) and methylation sequencing (MS) were performed with methylation specific primers. Results: Methyltransfected human umbilical cord blood samples were positive for MSP, the sequence of which was consistent with the expectation and negative for the untransferred methyl groups. The positive rates of APC methylation in tumor and normal lung tissue of 17 patients with lung cancer were 47% (8/17) and 18% (3/17), respectively. One case of benign lung tumor and its normal lung tissue The results were negative. The tumor and normal lung tissues of 9 lung cancer patients with MSP negative were detected by MS. There were 4 cases of CpG methylation in APC promoter. Conclusion: Using MSP and MS methylation detection and analysis, the positive rate of detection of 17 cases of clinically diagnosed lung cancer tissues was 71% (12/17). MSP is simple, inexpensive and suitable for screening samples of large-scale oncology patients. For MSP-negative clinical samples, MS can be used for further confirmation to provide more accurate information for the clinical diagnosis of lung cancer.