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目的:建立一种可检测微量DNA标本中DNA甲基化的甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzymes-based quantitative PCR,MSRE-qPCR)方法,并运用该技术探讨血浆Ras相关区域家族蛋白1A(Ras association domain family1A,RASSF1A)基因甲基化检测在肝细胞癌(hepatocellutar carcinoma,HCC)非侵入性诊断中的价值。方法:用MSRE HhaⅠ消化DNA样品,再用qPCR技术分析酶切结果,建立检测RASSF1A基因甲基化的MSRE-qPCR方法。以45例肝组织(20对HCC患者手术切除肿瘤标本及匹配非癌组织和5例正常肝)为材料,测试该方法的应用价值;运用亚硫酸氢盐测序PCR(bisulf ite sequencing PCR,BSP)技术进行进一步验证,并与甲基化特异性PCR(methylation specif ic PCR,MSP)方法相比较。再运用该技术检测150例血浆标本(包括72例HCC患者、37例肝硬化或慢性肝炎等良性病变患者和41例健康对照)的RASSF1A基因甲基化状态,并分析其与HCC患者临床病理参数的关系。结果:MSRE-qPCR法可定量检测低至1%以下的RASSF1A甲基化片段。20例HCC组织中有14例(70%)发生RASSF1A高甲基化,对应非癌组织中RASSF1A甲基化阳性率为25%,而5例正常肝组织均为阴性。MSRE-qPCR结果经BSP验证无误,且与MSP检测结果具有较好的一致性。HCC患者血浆RASSF1A甲基化阳性率(47/72,65.3%)显著高于健康对照(1/41,2.4%)和肝良性病变组(3/37,8.1%),差异均有统计学意义(P<0.0001)。联合检测血浆RASSF1A甲基化与血清AFP可显著提高HCC诊断效率。结论:建立的MSRE-qPCR方法要求样本少、操作简便、成本低廉,可定量检测RASSF1A基因甲基化水平。血浆RASSF1A甲基化分析对于HCC的非侵入性诊断具有重要价值。
OBJECTIVE: To establish a methylation-sensitive restriction enzymes-based quantitative PCR (MSRE-qPCR) method for detecting DNA methylation in DNA samples. With this technique To investigate the value of methylation detection of Ras association domain family1A (RASSF1A) gene in the noninvasive diagnosis of hepatocellular carcinoma (HCC). METHODS: DNA samples were digested with MSRE Hha I and analyzed by qPCR. The MSRE-qPCR method was established to detect the methylation of RASSF1A gene. The application value of this method was tested in 45 cases of liver tissues (20 specimens of HCC with resected tumor, non-cancerous tissues and 5 normal liver tissues). By bisulfite sequencing PCR (BSP) The technique was further validated and compared with the methylation specif ic PCR (MSP) method. The methylation status of RASSF1A gene in 150 plasma samples (including 72 HCC patients, 37 benign lesions of liver cirrhosis or chronic hepatitis and 41 healthy controls) was detected by this technique. The methylation status of RASSF1A gene in HCC patients was analyzed. Relationship. Results: The MSRE-qPCR assay quantifies RASSF1A methylated fragments down to 1%. RASSF1A hypermethylation occurred in 14 of 20 (70%) HCC tissues, corresponding to a 25% positive rate of RASSF1A methylation in non-cancerous tissues, whereas all 5 normal liver tissues were negative. The results of MSRE-qPCR were verified by BSP and had good consistency with MSP test results. The positive rate of RASSF1A methylation in plasma of HCC patients was significantly higher than that of healthy controls (1 / 41,2.4%) and benign liver lesions (3 / 37,8.1%) (47/72, 65.3%), the differences were statistically significant (P <0.0001). Combined detection of plasma RASSF1A methylation and serum AFP can significantly improve the diagnostic efficiency of HCC. Conclusion: The established MSRE-qPCR method requires less sample, simple and convenient operation and low cost, and can quantitatively detect the methylation level of RASSF1A gene. Plasma RASSF1A methylation analysis is of great value in the noninvasive diagnosis of HCC.