激光捕获显微切割联合SELDI蛋白质芯片筛选肺腺癌早期诊断标志蛋白  

Application of Laser Capture Microdissection and Surface-enhanced Laser Desorption Ionization Time-of-flight Mass Spectrometry to Screen Biomarkers for Early Diagnosis of Lung Adenocarcinoma

作　　者：田应选[1] 杨拴盈[1] 南岩东[1] 张潍[1] 余捷凯[2] 郑树[2] 

机构地区：[1]西安交通大学医学院第二附属医院呼吸科,西安710004 [2]浙江大学医学院附属第二医院肿瘤研究所

出　　处：《四川大学学报（医学版）》2009年第1期157-161,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30570795);教育部新世纪优秀人才支持计划项目(NECT-06-0845)资助

摘　　要：目的探讨应用激光显微切割(LCM)联合表面增强激光解吸离子化飞行时间质谱蛋白质芯片(SEL-DI-TOF-MS)及模式识别分类技术—支持向量机(SVM)筛选肺腺癌标志蛋白的可行性。方法将6例新鲜肺腺癌组织标本及其配对的4例正常肺组织制备8μm厚度冰冻切片;改良HE染色;用LCM技术选择性获取同质腺癌细胞和配对正常肺组织细胞。应用PBSⅡ+型SELDI-TOF-MS分析仪(IMAC芯片)分析腺癌及其配对正常细胞的蛋白质表达谱,比对差异点;应用SVM筛选并验证候选标志蛋白的判别效能。结果平均每个LCM帽子的激光点数约4000shots,获得了同质性>95%的肿瘤细胞和正常细胞。比较腺癌和配对正常细胞之间的SELDI谱图,共筛选出84个蛋白峰。将差异最明显的10个蛋白峰作为候选标志蛋白。和正常组织相比,6种蛋白在腺癌中呈低表达,4种蛋白在腺癌中呈高表达,差异有统计学意义(P<0.05)。初步筛选出3191m/z蛋白峰作为腺癌诊断标志蛋白。结论LCM联合SELDI蛋白质芯片技术有可能筛选出敏感性高、特异性强的肺腺癌标志蛋白;该技术将为肺腺癌早期诊断研究提供新的强有力的工具。Objective To improve diagnostic methods to screen biomarkers for early diagnosis in lung adenocarcinoma by employing laser capture microdissection （LCM） and surface-enhanced laser desorption ionization time- of-flight mass spectrometry （SELDI-TOF-MS） and support vector machine （SVM）. Methods Frozen sections of thickness 8 μm were made using 6 cases of fresh lung cancer tissues and 4 cases of matched normal lung tissues. The sections were stained by improved HE solution. The homogeneous adenocarcinoma cells and normal cells were collected by LCM in each sample, and then SELDI profiles based on PBS Ⅱ^ ＋ SELDI-TOF-MS （IMAC protein chip） were analyzed using SVM. Results High quality cell samples were obtained by LCM quickly and precisely from normal specimens and diseased tissues without interstitium, inflammation and necrosis. Eighty four differential protein peaks were found. Top ten of them were identified as candidate biomarkers. six proteins were significantly weakly expressed in lung cancer tissue compared to normal tissues, but the other four protein were over-expressed （P〈0. 05）. Every candidate biomarker has undergone the blind-cross-test. Each of them can separate the lung cancer from normal samples with a sensitivity of 100% and a specificity of 100%. The 3191 m/z was considered as disease marker of lung adenocarcinoma. Conclusion The method combined LCM with SELDI-TOF-MS may be able to screen potential biomarkers to distinguish lung cancer from healthy tissue with high sensitivity and specificity, which could improve early diagnosis for lung cancer.

关 键 词：激光捕获显微切割 表面增强激光解吸离子化飞行时间质谱 蛋白质组学 肺腺癌 早期诊断 

分 类 号：R734.2[医药卫生—肿瘤]