肝癌相关基因表达的荧光定量PCR检测及分子诊断指数的建立  

Detection of HCC-associated gene expression by real-time fluorescence quantitative PCR to establish a molecular diagnostic index for HCC

作　　者：余俊[1] 王毅军[2] 高英堂[3] 石文霞[4] 王瞿辉[3] 刘彤[4] 许彦杰[3] 杨斌[3] 杜智[3] 

机构地区：[1]天津医科大学第三中心临床学院,天津市300170 [2]天津市第三中心医院肝胆外科,天津市300170 [3]天津市第三中心医院天津市人工细胞重点实验室,天津市300170 [4]天津市第三中心医院天津市肝胆疾病研究所,天津市300170

出　　处：《世界华人消化杂志》2011年第6期588-595,共8页World Chinese Journal of Digestology

基　　金：天津市重点科技攻关基金资助项目;No. 05YFSZSF02500;天津市卫生局基金面上资助项目;No. 09KY04~~

摘　　要：目的:研究肝细胞癌相关基因表达,建立肝癌分子诊断指数,以期能更准确的诊断肝癌.方法:采用实时荧光定量PCR检测40例肝细胞癌患者癌组织和配对的癌旁2cm及手术切缘组织、10例肝硬化组织、10例正常肝脏组织中11个基因的表达,以管家基因G3PDH为对照,2-△△CT法计算目的基因相对表达量,挑选出特异性好且与正常肝、肝硬化组织表达差异大的肝癌相关基因,依据这些基因在组织中表达异常的个数建立分子诊断指数.结果:在40例癌组织中,抑癌基因PRDM2、IGFBP3、DLC-1分别有65.0%、75.0%、67.5%的标本表达<3倍正常肝组织,癌基因GPC3、STMN、CCNA2、BIRC5、AFP分别有87.5%、77.5%、82.5%、85.0%、67.5%的标本表达>3倍正常肝组织,并且与肝硬化组织表达差异显著(0.45±0.69vs0.50±0.20;0.17±0.20vs0.67±0.47;0.29±0.48vs0.58±0.60;677.57±999.30vs4.41±3.99;17.56±28.28vs1.17±1.08;53.17±103.64vs2.09±1.50;16.53±16.39vs1.82±1.39;4445.70±11642.87vs0.86±0.43,均P<0.05).以此8个基因建立分子诊断指数,其在肝硬化、手术切缘、癌旁2cm、癌组织中分别为2.2±1.5、3.0±1.6、2.9±1.5、6.3±1.2,癌组与肝硬化组、切缘组、癌旁2cm组差异显著.以肝硬化组作为对照组,当分子诊断指数≥4诊断为肝癌时,ROC曲线的敏感性为100%,特异性为90%,AUC=0.995.结论:利用荧光定量PCR检测肝癌相关基因表达,成功建立分子诊断指数,提高了肝癌诊断的敏感性及特异性.AIM：To develop a molecular diagnostic index for hepatocellular carcinoma（HCC）.METHODS：The expression of 11 genes was assessed by real-time fluorescent quantitative polymerase chain reaction（QRT-PCR） in 40 HCC specimens and matched tumor-adjacent specimens and surgical margin specimens,10 cirrhotic tissue specimens,and 10 normal liver tissue specimens.Using G3PDH as a control,the 2-△△CT method was used to calculate the relative gene expression levels.HCC-associated genes were then selected to establish a molecular diagnostic model for HCC.RESULTS：Compared with normal liver tissue specimens,approximately 65.0%,75.0%,and 67.5% of HCC specimens showed a 3-fold increase in the expression levels of tumor suppressor genes PRDM2,IGFBP3,and DLC-1,and approximately 87.5%,77.5%,82.5%,85.0%,and 67.5% of HCC specimens showed a 3-fold decrease in the expression levels of oncogenes GPC3,STMN,CCNA2,BIRC5,and AFP,respectively.The expression levels of these eight genes differed signif icantly between HCC and cirrhotic tissue（0.45 ± 0.69 vs 0.50 ± 0.20;0.17 ± 0.20 vs 0.67 ± 0.47;0.29 ± 0.48 vs 0.58 ± 0.60;677.57 ± 999.30 vs 4.41 ± 3.99;17.56 ± 28.28 vs 1.17 ± 1.08;53.17 ± 103.64 vs 2.09 ± 1.50;16.53 ± 16.39 vs 1.82 ± 1.39;4445.70 ± 11642.87 vs 0.86 ± 0.43,all P 0.05）.Molecular diagnostic index was estimated based on these eight genes,which was 2.2 ± 1.5,3.0 ± 1.6,2.9 ± 1.5,6.3 ± 1.2 for liver cirrhosis,surgical margin,tumor-adjacent tissue,and cancer tissue,respectively.The molecular diagnostic index for cancer tissue was significantly different from those for liver cirrhosis,surgical margin,and tumor-adjacent tissue.When a molecular diagnostic index of 4 or greater was adopted to diagnose liver cancer using liver cirrhosis as a control,the sensitivity,specificity,and the area under the receiving operative curve（ROC） were 100%,90%,and 0.995,respectively.CONCLUSION：A molecular diagnostic index for HCC was successfully established using fluorescence quantitative PCR to detect HCCassocia

关 键 词：肝细胞癌 基因 表达 实时荧光定量PCR 分子诊断指数 

分 类 号：R735.7[医药卫生—肿瘤]