NLCM纯化的不同分化程度贲门腺癌细胞差异蛋白质组学研究  

Differential proteomics analysis of navigated laser capture microdissection of well and poor differentiated human gastric cardia adenocarcinoma

作　　者：程妍[1] 李阳[2] 刘冬[1] 宋亚华[1] 张军[1] 龚均[1] 

机构地区：[1]西安交通大学医学部第二附属医院消化内科,陕西西安710004 [2]西安交通大学医学部第二附属医院耳鼻咽喉头颈外科,陕西西安710004

出　　处：《西安交通大学学报（医学版）》2015年第5期682-686,714,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30470785)~~

摘　　要：目的联合导向激光捕获显微切割技术(NLCM)及蛋白质组学技术比较分析高分化贲门腺癌细胞和低分化贲门腺癌细胞的蛋白质表达图谱差异,寻找与贲门腺癌恶性程度和预后有关的分子标志物。方法应用导向激光捕获显微切割技术分别获取高分化和低分化贲门腺癌细胞,提取蛋白质,应用双向凝胶电泳(2-DE)技术分离蛋白质,选定差异点,胶内酶解后,用基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)鉴定差异蛋白质,并用免疫组化方法验证差异蛋白的表达。结果 1NLCM分别分离纯化了高分化和低分化贲门腺癌细胞;2建立了高分化和低分化贲门腺癌细胞的蛋白质表达图谱,两组分别检测到(846±52)个和(923±84)个蛋白质点,两组的匹配率为85.4%;3对高分化和低分化的贲门腺癌蛋白质表达谱进行比较分析,鉴定出10种差异蛋白,其中6个蛋白质在低分化贲门腺癌细胞中表达上调,4个蛋白质在低分化贲门腺癌细胞中表达下调;4差异蛋白质涉及细胞间信号转导、细胞代谢、凋亡及迁移等;5进一步通过免疫组化方法验证了差异蛋白HSP27的表达与蛋白质组学研究结果一致。结论高分化和低分化贲门腺癌间存在蛋白质表达的差异,这些差异表达的蛋白质可能与贲门腺癌的恶性程度和预后有关。Objective To obtain protein biomarkers used for malignant degree and prognosis of human gastric cardiac adenocarcinoma（GCA）tissues captured by navigated laser capture microdissection（NLCM）and proteomics technology.Methods We performed navigated LCM to enrich the malignant gastric cardiac cells from surgical specimens of human GCA.The proteins extracted from these cells were then separated.Differential protein spots were identified by peptide mass fingerprint（PMF）based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS）and database searching.To validate the expression patterns of HSP27,immunohistochemistry was performed using formalin-fixed and paraffin-embedded tissue specimens.Results1 We obtained the 2-DE patterns of high resolution and reproducibility of well（group A）and poorly（group B）differentiated human GCA tissues captured by NLCM.2 The detected spots between them were as follows：846±52 and923±84 between group A and group B.The percentage of matched spots between them was 85.4%.3 With mass spectrometry technology（peptide mass fingerprint）,10 protein spots were successfully identified.Six proteins had significantly higher expression in poorly differentiated GCA than in well differentiated GCA.Another 4proteins had a significantly lower expression in poorly differentiated GCA than in well differentiated GCA.4 The identified proteins of GCA were classified in terms of their cellular localization and physiological function using information from SWISS-PROT and NCBI websites.Most of the proteins were involved in cell signal transduction,cell metabolism,apoptosis and migration.5 The expression of HSP27 in well differentiated GCA was higher than in poorly differentiated GCA group（P0.05）,which was in agreement with proteomic results.Conclusion Ten differentially expressed proteins identified between the poorly differentiated and well differentiated GCA tissues indicate that they may be associated with the malignance degree of GCA and the

关 键 词：贲门腺癌 激光捕获显微切割 蛋白质组学 分子标志物 

分 类 号：R735.2[医药卫生—肿瘤]