CAJ | 학술논문

目的建立一种包含甲基化标准品的新型高通量肺癌相关基因的甲基化检测系统.方法 2009年3-6月上海交通大学医学院附属胸科医院经病理学和影像学确认的非小细胞肺癌患者8例,其中男7例,女1例,平均年龄(57±11)岁,手术后留取肺癌组织及外周血.男性健康志愿者1名,年龄35岁,抽取外周血20 ml,用以分离单个核细胞提取DNA制备质粒.对7个肺癌相关基因的启动区进行克隆,用各基因的特异性引物对克隆到质粒中的各基因片段再次进行扩增,PCR产物用甲基化酶M.sss Ⅰ和亚硫酸盐处理.通过设计3'末端CpGpCpG探针来识别甲基化模板,并经最佳连接酶、最佳退火温度及连接温度的选择,建立一套多重连接PCR体系,最后运用液相芯片平台对扩增的标准品和临床标本进行检测.利用甲基化特异性PCR检测新系统的可靠性.结果成功的构建了7个基因的甲基化和非甲基化标准品,并建立了多重PCR偶联液相芯片的快速检测系统,p16INK4A、APC、DAPK、RARβ、RASSF1 A、MGMT和GSTP1甲基化标准品的荧光信号值分别为863、909、703、701、901、1 060和885,与非甲基化标准品区分明显.抽提8例患者的肺癌组织DNA,对其中检测出阳性频率较高的基因进行甲基化特异性PCR,产物电泳结果与新建甲基化检测系统检测结果完全一致.结论本研究建立的新型检测系统能同时准确地检测7个基因甲基化状态,有望应用于临床标本的检测.
목적 건립일충포함갑기화표준품적신형고통량폐암상관기인적갑기화검측계통.방법 2009년3-6월상해교통대학의학원부속흉과의원경병이학화영상학학인적비소세포폐암환자8례,기중남7례,녀1례,평균년령(57±11)세,수술후류취폐암조직급외주혈.남성건강지원자1명,년령35세,추취외주혈20 ml,용이분리단개핵세포제취DNA제비질립.대7개폐암상관기인적계동구진행극륭,용각기인적특이성인물대극륭도질립중적각기인편단재차진행확증,PCR산물용갑기화매M.sss Ⅰ화아류산염처리.통과설계3'말단CpGpCpG탐침래식별갑기화모판,병경최가련접매、최가퇴화온도급련접온도적선택,건립일투다중련접PCR체계,최후운용액상심편평태대확증적표준품화림상표본진행검측.이용갑기화특이성PCR검측신계통적가고성.결과 성공적구건료7개기인적갑기화화비갑기화표준품,병건립료다중PCR우련액상심편적쾌속검측계통,p16INK4A、APC、DAPK、RARβ、RASSF1 A、MGMT화GSTP1갑기화표준품적형광신호치분별위863、909、703、701、901、1 060화885,여비갑기화표준품구분명현.추제8례환자적폐암조직DNA,대기중검측출양성빈솔교고적기인진행갑기화특이성PCR,산물전영결과 여신건갑기화검측계통검측결과 완전일치.결론 본연구건립적신형검측계통능동시준학지검측7개기인갑기화상태,유망응용우림상표본적검측.
Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.