蛇毒半胱氨酸蛋白酶抑制剂基因转染对小鼠黑色素瘤B16细胞蛋白质表达谱的影响  

Effect of snake venom cystatin gene on the protein expression profiles of mouse melanoma B16F1 cells

作　　者：齐元麟[1] 纪明开[1,2] 谢群[1,3] 黄清玲[1] 林建银[1] 

机构地区：[1]福建医科大学基础医学院分子医学研究中心,消化道恶性肿瘤教育部重点实验室,福建福州350004 [2]福建医科大学附属第一医院皮肤科,福建福州350004 [3]莆田学院医学院,福建莆田351100

出　　处：《中国药理学通报》2011年第6期791-797,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30371747);福建省教育厅资助省属高校项目(No2007F5051);福建医科大学青年科学基金资助项目(NoFJGXQ04011)

摘　　要：目的研究蛇毒半胱氨酸蛋白酶抑制剂(sv-cystatin)基因转染对小鼠黑素瘤细胞B16F1的蛋白表达谱的影响,探讨其抗肿瘤机制。方法采用双向电泳-质谱的蛋白质组学研究策略,分析稳定表达sv-cystatin的B16F1细胞系B16F1/cystatin与转染空载体的对照细胞B16F1/pcDNA3.1的蛋白表达谱的差别,筛选鉴定差异表达蛋白;用实时定量PCR和Western blot进一步确定实验结果;对蛋白组数据和前期基因组数据进行比较分析。结果 B16F1/cystatin和B16F1/pcDNA3.1细胞总蛋白的双向电泳图谱上共有匹配蛋白点(412±20)个,其中有11个蛋白的表达差异在2倍以上,5个上调,6个下调。这11个蛋白和1个仅在B16F1/cystatin中表达的蛋白经MALDI-TOF-MS分析,其中10个蛋白得到了鉴定,它们分属于代谢酶系、小G蛋白Ran调控分子、真核翻译因子、核苷酸激酶等。Western blot显示NM23和RhoGDI-beta的蛋白表达分别上调了(2.23±0.34)倍和(1.57±0.11)倍;实时定量PCR结果显示NM23、RhoGDI-beta、RANBP1的mRNA表达上调,eIF5A、eEF1-beta2、MDH1表达下调;与蛋白质组学研究结果一致。基于Gene Ontology的分析提示sv-cystatin作用的靶分子主要定位于细胞外周、质膜和细胞核,与转录、半胱氨酸蛋白酶活性、细胞凋亡等过程有关。结论 sv-cystatin可能通过调控抑癌基因NM23、RhoGDI等蛋白质发挥其抗肿瘤作用。sv-cystatin的主要靶分子可能是转录因子、分泌蛋白和质膜受体。Aim To investigate the effects of stable transfected sv-cystatin gene on the protein expression profiles of mouse melanoma B16F1 cells.Methods By using two-dimensional gel electrophoresis followed by MALDI-TOF-MS and database searching,the differential protein expression profile between the B16F1/sv-cystatin cells which stably expressed the sv-cystatin gene and the B16F1/pcDNA3.1 cells which was transfected with the control vector pcDNA3.1-His C was analysed.The mRNA and protein expressions of some differentially expressed proteins were confirmed by real-time PCR and Western blot.The differentially expression genes were analyzed by a web-based DAVID bioinformatics platform.Results Out of（412±20） matched spots on two-dimensional gels,11 spots with altered expressions were found,five were up-regulated and six were down-regulated.Ten proteins were identified by MALDI-TOF-MS.These altered proteins were involved in cell metabolism,small G-protein regula-tion,translation,etc.The results of Western blot showed that the expressions of tumor suppressor gene NM23 and the Ran regulator RhoGDI-beta were up-regulated by（2.23±0.34）and（1.57±0.11）folds,respectively.The mRNA expressions of NM23,RhoG-DI-beta and RANBP1 were up-regulated whereas the expressions of eIF5A,eEF1-beta2 and MDH1 were down-regulated.These results were in accordance with the data from proteomic analysis.Analysis based on the gene ontology annotation suggested the target genes of sv-cystatin mainly located in extracellular region,plasma membrane and nucleus,and the functions of these genes involved regulation of transcription,cysteine proteases and apoptosis.Conclusions The regulation of NM23 and RhoGDI might contribute to the antitumor activities of sv-cystatin.The targets of sv-cysta-tin might be transcriptional factors,secreted enzymes and receptors.

关 键 词：蛇毒 半胱氨酸蛋白酶抑制剂 黑色素瘤 小鼠 蛋白质组学 基因本体学 

分 类 号：R-332[医药卫生] R394.2