摘要
目的：本研究的目的在于通过检测肺癌患者癌组织、相应的支气管灌洗液(Bronchoalveolar Lavage Fluid, BALF)以及远癌肺组织和非肿瘤患者的支气管灌洗液的甲基化水平,初步筛选并建立用于肺癌早期诊断的DNA甲基化分子标志物。
方法：1.2008年10月-2009年10月天津医科大学总医院肺外科手术治疗肺癌患者15例,留取患者的肺部肿瘤标本、远癌肺组织及支气管灌洗液标本。应用甲基化特异性PCR的方法对各组标本进行甲基化检测,判断RARβ、DAPK、MGMT、APClA、ECAD5个基因的甲基化水平,筛选出用于肺癌早期诊断的基因。2.2008年10月-2009年10月天津医科大学总医院最终经病理确诊为肺癌的患者69例作为病例组,肺部良性病变患者30例作为对照组,留取支BALF,应用甲基化特异性PCR的方法进行甲基化检测,判断RARβ、DAPK、MGMT APClA、ECAD 5个基因的甲基化水平,分析DNA甲基化与肺癌发生的关系。
结果：(1)肺癌组织：RARβ、DAPK、MGMT、APClA、ECAD及多基因联合检测启动子甲基化检出率分别为66.7%(10/15)、73.3%(11/15)、53.3%(8/15)、80%(12/15)、66.7%(10/15)和100%(15/15)；
(2)远癌肺组织：RARβ、DAPK、MGMT、APClA、ECAD及多基因联合检测启动子甲基化检出率分别为53.3%(8/15)、0%(0/15)、26.7%(4/15)、73.3%(11/15)、0%(0/15)和93.3%(14/15)；
(3)支气管灌洗液：RARβ、DAPK、MGMT、APClA、ECAD及多基因联合检测启动子甲基化检出率分别为73.3%(11/15)、46.7%(7/15)、80%(12/15)、40%(6/15)、20%(3/15)；和100%(15/15)；
(4)支气管灌洗液中肺癌组与良性病变组甲基化发生率比较：RARβ和APClA基因甲基化发生率在两组中区别有统计学意义(P=0.00,P=0.041);
(5)肺癌诊断的敏感性和特异性：RARβ基因诊断肺癌的敏感性为60.9%特异性为80%；DAPK基因诊断肺癌的敏感性为56.5%特异性为53.3%; MGMT基因诊断肺癌的敏感性为50.7%特异性为56.7%; APClA基因诊断肺癌的敏感性为42%特异性为80%；ECAD基因诊断肺癌的敏感性为10.1%特异性为83%；多基因联合检测断肺癌的敏感性为91.3%和23.3%；
(6)抑癌基因启动子甲基化与肺癌相关性：经多因素回归分析发现,高龄及RARβ基因启动子甲基为肺癌发生的危险因素OR值分别为7.565(1.687～33.922)和6.833(1.937～24.108)。
结论：(1)肺癌患者癌组织、远癌组织和支气管灌洗液中均存在不同程度的RARβ、DAPK、MGMT、APClA和ECAD基因启动子过度甲基化；(2)肺癌患者癌组织与支气管灌洗液中抑癌基因启动子过度甲基化水平相似；(3)多基因联合检测支气管灌洗液中启动子甲基化有助于肺癌早期诊断；(4)单独检测某一个抑癌基因启动子甲基化作为肺癌诊断的分子标志物敏感性和特异性不高；(5)多基因联合检测可以提高肺癌诊断的敏感性。
OBJECTIVE:The aim of this research was to find the candidate genes for lung cancer early diagnosis by using DNA specific PCR (MSP).
METHODS:We selected 15 patients with lung cancer in the pulmonary surgery department of the Tianjin medical university general hospital from October 2008 to October 2009 and getted the tumor tissue the tumor remote lung tissue and BALF of the same patients. We extracted DNA from the samples and applied MSP which based on sodium bisulfite modification to detect the methylation status between the three different group samples. We detected RARβ、DAPK、MGMT、APClA、ECAD gene methylation status to find the difference between the three-group samples. 2.69 patients with lung cancer and 30 non-tumor patients was selected in the pulmonary surgery and respiratory department of general hospital from October 2008 to October 2009 the lung cancer patients was defined as cases and the non-tumor patients as control. We detected RARβ、DAPK、MGMT、APClA.ECAD gene methylation statu difference by using MSP in the two groups in the BALF and analyzed the relationship between the two groups in associated with the 5 genes methylation status.
RESULTS:(1)In the lung cancer tissue,the methylation positive rate of the RARβ、DAPK、MGMT、APClA,ECAD and multiple genes was 53.3%(8/15)、0%(0/15)、26.7%(4/15)、73.3%(11/15)、0%(0/15) and 93.3%(14/15) respectively;
(2)In the remote lung tumor tissue,the methylation positive rate of the RARβ、DAPK、MGMT、APClA、ECAD and multiple genes was 53.3%(8/15)、0%(0/15)、26.7%(4/15)、73.3%(11/15)、0%(0/15)and 93.3%(14/15);
(3)In the BALF,the methylation positive rate of the RARβ、DAPK、MGMT、APCIA、ECAD and multiple genes was 73.3%(11/15)、46.7%(7/15)、80%(12/15)、40%(6/15)、20%(3/15)and100%(15/15);
(4)The methylation status of the RARβand APClA reached statistical difference (P=0.00, P=0.041) between lung cancer group and non-malignant tumor group;
(5)The sensitivity and specificity of DNA methylation to predict lung cancer:The sensitivity of the 5 genes were 60.0%,56.5%,50.7%,42%,10.1%; and the specificity were 80%,53%,56.7%,80%,83% respectivelly;
(6)DNA methylation and lung cancer risk.By using logistic regression,we found advanced age and RARβgene promoter hypermethylation were risk factor of lung cancer with an odds ratio of 7.565 (1.687～33.922) and 6.833 (1.937～24.108) respectively.
CONCLUSIONS:(1)Hypermetylation of the RARβ、DAPK、MGMT、APClA、ECAD five genes was observed in lung cancer tissue remote lung tumor tissue and BALF of the lung cancer patients.(2)The hypermethylation status of the tumor inhibite genes was similar between lung cancer tissue and BALF.(3)It was usefull for lung cancer early diagnosis by detecting multiple gene methylation status.(4)Sensitivity and specificity was not acceptable by using single gene methylation status.(5)The sensitivity and specificity was improved by detecting multiple tumor inhibitor genes.
引文
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