【摘要】 目的:建立甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzyme-quantitative PCR,MSRE-qPCR)方法,并应用该方法探讨血浆腺瘤性结肠息肉病(adenomatous polyposis coli,APC)基因甲基化检测在肝细胞癌(hepatocellular carcinoma,HCC)诊断中的应用价值。方法:用HhaⅠ消化DNA样品,qPCR技术评估酶切效率,建立优化的MSRE-qPCR方法。用MSRE-qPCR法检测45例肝组织(20例HCC及其相应匹配的非癌组织和5例正常肝组织)中APC甲基化水平;应用亚硫酸氢盐测序PCR(bisulfi te sequencing PCR,BSP)对MSRE-qPCR检测结果进行验证,并与甲基化特异性PCR(methylation-specifi c PCR,MSP)检测方法比较。运用MSRE-qPCR技术检测72例HCC、37例肝良性病变和41例健康志愿者血浆标本的APC甲基化状态。结果:建立的MSRE-qPCR方法可定量检测低至1%的APC甲基化片段。MSRE-qPCR和MSP检测结果均显示,HCC组织中APC基因发生高频率甲基化。MSRE-qPCR检测结果经BSP验证准确无误,且与MSP检测结果具有较好的一致性(Kappa=0.955,P<0.0001)。HCC患者血浆APC甲基化水平高于肝良性病变及健康志愿者(P<0.0001)。血浆APC甲基化分析与血清甲胎蛋白(alpha-fetoprotein,AFP)联合检测可提高HCC诊断效率。结论:MSRE-qPCR可定量检测APC甲基化水平。血浆APC甲基化分析对于HCC的非侵入性诊断具有重要价值。更多还原

【Abstract】 Objective:To establish a method of methylation-sensitive restriction enzymes-quantitative PCR(MSRE-qPCR) for methylation analysis of adenomatous polyposis coli(APC) gene,and to further assess the clinical value of plasma methylation detection by using this method in diagnosis of hepatocellular carcinoma(HCC).Methods:Hha I was used to digest genomic DNA,and the digestion efficiency was evaluated by using qPCR technique.Then the optimized MSRE-qPCR method was established.The methylation levels of APC in 45 liver tissues(20 surgically resected HCC specimens and the matched non-cancerous tissues,as well as 5 normal liver tissues) were detected by MSRE-qPCR,and then further validated by using bisulfite sequencing PCR(BSP).The results of MSRE-qPCR were compared with those of methylation-specific PCR(MSP) assay.MSRE-qPCR method was used to detect the APC methylation status of plasma samples from 72 cases of HCC,37 cases of benign liver diseases and 41 healthy volunteers.Results:The established MSRE-qPCR method could detect as low as 1% methylated target sites in given DNA samples.The results of MSRE-qPCR and MSP showed that APC gene was hypermethylated in HCC tissues.The result of MSRE-qPCR was verified by BSP,and it was comparable with that of MSP(Kappa=0.955,P<0.000 1).Methylation level of plasma APC in patients with HCC was significant higher than those in patients with benign liver diseases and the healthy volunteers(P<0.000 1).Combined analysis of plasma APC methylation and serum alpha-fetoprotein(AFP) revealed an increased diagnostic efficacy for HCC.Conclusion:MSRE-qPCR is a method for quantitative analysis of APC methylation level.Methylation analysis of plasma APC is a valuable method for the noninvasive diagnosis of HCC.更多还原