激光捕获显微切割技术纯化的鼻咽癌和正常鼻咽上皮细胞的比较蛋白质组学研究  

Comparative Proteomics Analysis of LCM Purified Nasopharyngeal Carcinoma and Normal Nasopharyngeal Epithelial Tissue

作　　者：程爱兰[1] 黄卫国[1] 张鹏飞[1] 李茂玉[1] 彭芳[1] 李峰[1] 李萃[1] 易红[1] 李美香[1] 陈主初[1] 肖志强[1] 

机构地区：[1]中南大学湘雅医院医学实验研究中心,卫生部肿瘤蛋白质组学重点实验室,长沙410008

出　　处：《生物化学与生物物理进展》2008年第5期521-530,共10页Progress In Biochemistry and Biophysics

基　　金：国家重点基础研究发展计划(973)(2001CB5120);芙蓉学者特聘教授科研奖励基金(湘教通[2007]362号);教育部跨世纪优秀人才培养计划基金(教计函[2002]48);湖南省科技厅重大科技专项(04XK1001,06SK2004)资助项目~~

摘　　要：为筛选鼻咽癌(nasopharyngeal carcinoma,NPC)的分子标志物,采用激光捕获显微切割技术(laser capture microdissection,LCM)分别从NPC组织和正常鼻咽上皮组织(normal nasopharyngal epithelial tissue,NNET)中切割纯化NPC细胞和正常鼻咽上皮细胞(normal nasopharyngal epithelial cells,NNEC),应用二维凝胶电泳(two-dimensional electrophoresis,2-DE)分离LCM纯化细胞的蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾电离串联质谱(ESI-Q-TOF-MS)鉴定差异蛋白质点,Western blot检测差异蛋白cytokeratin8(CK8)在LCM纯化的NPC细胞和NNEC以及具有不同分化程度或转移潜能的4株NPC细胞中的表达,免疫组织化学检测CK8在63例NPC、28例NNET及20例颈淋巴结转移NPC组织中的表达水平.建立了LCM纯化的NPC细胞和NNEC的2-DE图谱,质谱鉴定了29个差异蛋白质,其中15个蛋白质只在NPC表达或表达明显增高,14个蛋白质在NPC中表达下调或缺失;Western blot结果显示,CK8的表达水平在NPC中较NNET明显下调,并与NPC细胞株的分化程度和转移潜能有关;免疫组织化学结果显示,CK8在NPC组织中的表达较NNET明显下调,在颈淋巴结转移NPC中的表达较原发NPC明显上调.研究结果提示,CK8与NPC分化及淋巴结转移相关,有望成为预测NPC转移和区别NPC分化程度的分子标志物.To search for nasopharyngeal carcinoma （NPC） biomarkers, laser capture microdissection （LCM） was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues （NNET）. Two-dimensional gel electrophoresis （2-DE） was performed to separate the total proteins of microdissected NPC and NNEC, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the two types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. The expression of cytokeratin 8（CK8）, one of the differential proteins, in the microdissected NPC and NNEC as well as 4 NPC cell lines with different differentiated degrees and/or metastatic potentials was detected by Western blot. Immunohistochemistry was also used to detect the expression of CK8 in paraffin-embedded tissues including 63 cases of primary NPC, 28 cases of NNET and 20 cases of cervical lymphonode metastasis. In the present study, 2-DE patterns of microdissected NPC and NNEC were established, and 29 differential proteins in the above two tissues were identified, of which 15 only expressed or up-regulated in N-PC and 14 only expressed or up-regulated in NNET. The expression level of differential protein CK8 between the NPC and NNET was selectively confirmed, and was found to be related to the differentiation and/or metastasis of NPC cell lines. Significant down-regulation of CK8 was observed in NPC compared with NNET, and significant up-regulation of CK8 was also observed in lymphonode metastasis compared with primary NPC. The data suggest that CK8 may be related to the differentiation and lymphonode metastasis of NPC, and may serve as molecular biomarkers for metastasis and differentiation of NPC.

关 键 词：激光捕获显微切割 鼻咽癌 蛋白质组学 二维凝胶电泳 基质辅助激光解吸电离飞行时间质谱 电喷雾电离串联质谱 Western blot 免疫组织化学 

分 类 号：R318.0[医药卫生—生物医学工程]