激光显微切割结合蛋白质组学技术分析DDAH-1在肝细胞癌表达的变化  

Laser capture microdissection combined with proteome analysis in determining differential expression of DDAH-1 in hepatocellular carcinoma

作　　者：王庆[1] 洪毅[2] 胡和平[1] 谈冶雄[2] 艾建华[2] 陆豪杰[3] 刘淑琴[2] 王红阳[2] 

机构地区：[1]第二军医大学东方肝胆外科医院消化内科 [2]第二军医大学东方肝胆外科医院生物信号转导中心 [3]复旦大学生物化学研究所

出　　处：《中国肿瘤生物治疗杂志》2007年第6期505-510,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.20527004)~~

摘　　要：目的:应用激光显微切割(laser capture microdissection,LCM)结合蛋白组学技术筛查肝细胞癌(hepatocellular carcinoma,HCC)组织及其癌旁组织的差异表达蛋白,分析二甲基精氨酸二甲胺水解酶1(dimethylarginine dimethylaminohydrolase1,DDAH-1)在肝细胞癌表达的变化。方法:选取2003-2006年间东方肝胆外科医院原发性肝细胞癌患者的手术切除标本40例。利用LCM技术分离捕获肝癌组织及癌旁组织的肝实质细胞,应用二维凝胶电泳技术(two dimensional gel electrophoresis,2-DE)筛选全部标本共同差异表达频率>80%、差异强度>3倍的蛋白质点;应用电喷雾串联质谱(electrospray ionisation tandem mass spectrometry,ESI-MS/MS)及基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)对差异蛋白点进行质谱鉴定分析;采用Western blotting及免疫组化对差异蛋白DDAH-1进行检测。结果:2-DE筛查获得在肝癌及癌旁组织差异表达的蛋白质点共20个,通过质谱鉴定获得12个蛋白质,其中5个在肝癌组织表达上调,7个下调;这12个蛋白质与细胞代谢、增殖、分化及信号调控相关,其中的DDAH-1是参与调控一氧化氮相关通路的重要酶。Western blotting检测显示,20例标本中16例DDAH-1表达明显升高;免疫组化检测证实,10例标本DDAH-1全部呈强阳性表达。结论:肝细胞癌组织细胞中筛查到12个差异表达蛋白质,其中的DDAH-1在肝癌组织中表达上调,其有可能在肝癌发生、发展过程中起重要作用。Objective： To investigate the differential expression of dimethylarginine dimethylaminohydrolase 1 （DDAH- 1 ） in hepatocellular carcinoma （HCC） and surrounding non-tumor tissues using laser capture microdissection （LCM） combined with proteome analysis. Methods： Forty surgical specimens of HCC were from patients who were treated in Eastern Hepatobiliary Surgery Hospital in 2003 - 2006. LCM was applied to isolate hepatic parenchymal cells of cancerous tissues and surrounding non-cancerous tissues. Two-dimensional electrophoresis （2-DE） was used to screen for the differen- tial expression proteins （the differential frequency 〉 80% and the intensity 〉3 times）. The differentially expressed proteins were identified by electrospray ionization-tandem mass spectrometry（ESI-MS/MS） and matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Western blotting and immunohistochemistry were used to analyze the differential expression of DDAH-1 in HCC. Results： Totally 20 protein spots were identified to be differentially expressed between HCC tissue cells and the non-tumor tissue cells. Using ESI-MS/MS and MALDI-TOF MS technology, 12 spots were identified by peptide mass fingerprint （PMF）, with 5 spots upregulated and 7 spots downregulated in HCC cells, including metabolism related proteins, cell signal proteins, binding proteins and so on. Among these proteins,DDAH-1 is a critical regulator of the nitrogen monoxidum （NO） signaling pathway. Western blotting showed that the expression of DDAH-1 was markedly increased in 16 of 20 HCC tissues. Immunohistochemistry showed that DDAH-1 was localized in the cytoplasm and was highly expressed in HCC tissues. Conclusion： Twelve differentially expressed proteins have been identified in HCC cells. DDAH-1 is overexpressed in HCC tissue, which may play a very important role in the mechanism of hepatocarcinogenesis.

关 键 词：肝细胞癌 激光捕获显微切割 蛋白质组学 差异表达蛋白 二甲基精氨酸二甲胺水解酶1 

分 类 号：R735.7[医药卫生—肿瘤] R730.2[医药卫生—临床医学]