应用蛋白质组学技术筛选鉴定锌指蛋白蛋白质组学技术筛选鉴定锌指.PDF

·360 · doi 10.3969/j.issn. 1673-4254.2015.03.09 J South Med Univ, 2015, 35(3): 360-364 基础研究 应用应用蛋白质组学技术筛选鉴定锌指蛋白蛋白质组学技术筛选鉴定锌指蛋白139139 调控的胃癌转移相调控的胃癌转移相 关蛋关蛋白质白质 李 勇，王力利，康爱文，范立侨，赵 群，檀碧波，郝英杰，刘庆伟 河北医科大学第四医院外三科，河北 石家庄 050011 摘要：目的 以胃癌SGC7901 细胞原位移裸鼠模型为对象，通过荧光双向差异凝胶电泳（2D-DIGE）结合液质联用（LC-MS）质谱 分析技术鉴定ZNF 139 调控的胃癌转移相关蛋白质。方法 合成针对ZNF 139 的小干扰RNA （ZNF 139-siRNA），以 ZNF 139-siRNA 转染人胃癌细胞株SGC7901 ，G418 筛选。以ZNF 139-siRNA 质粒转染的胃癌细胞、阴性质粒转染的胃癌细胞 及普通胃癌细胞分别进行裸鼠胃癌原位移植。造模成功后取出原位移植瘤及腹腔转移淋巴结。荧光双向差异凝胶电泳 （2D-DIGE）技术分别分离ZNF 139-siRNA 各组原位移植瘤及腹腔转移淋巴结蛋白质；选定差异点，胶内酶解后，液质联用 （LC-MS）质谱分析技术鉴定蛋白质。蛋白印迹（Western blot）技术验证差异蛋白质的表达。结果 转染ZNF 139-siRNA 质粒后 SGC7901 细胞中ZNF 139 的表达受到有效抑制。与阴性质粒组及空白组比较，阳性质粒组原位移植瘤生长更慢（P0.05），且腹 腔淋巴结转移率更低（P0.05）。蛋白质组学结果发现在阳性质粒组原发灶中Fascin、hnRNPA2/B 1表达下调，ANXA 1表达上 调；阳性质粒组转移淋巴结中ANXA5 表达下调（P0.05）。Western blot 验证结果与蛋白质组学结果相符。结论 ZNF 139可能 通过调节Fascin、hnRNPA2/B 1、ANXA 1、ANXA5促进胃癌淋巴结转移。 关键词：胃癌；锌指蛋白139；原位移植裸鼠；淋巴结转移；蛋白质组学 Screening and identification of proteins related to gastric cancer metastasis with comparative proteomics LI Yong, WANG Lili, KANG Aiwen, FAN Liqiao, ZHAO Qun, TAN Bibo, HAO Yingjie, LIU Qingwei Third Department of Surgery, Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang 050011, China Abstract: Objective To screen and identify the proteins related with tumor metastasis of gastric cancer in a nude mouse model bearing orthotopic transplanted tumor. Methods Zinc finger protein 139 (ZNF139)-specific siRNA was synthesized and transfected into gastric cancer cell line SGC7901, which was then screened by G418. ZNF139-siRNA-transfected cells, negative plasmid-transfected cells and untreated SGC7901 cells were orthotopically transplanted separately on the stomach wall of BALB/c nude mice. The primary tumors and metastatic lymph nodes were harvested to separate the proteins by 2-D