【作者】 曾希； 苏琦； 易岚； 唐海林； 廖爱军； 解娜； 刘亮；

【Author】 ZengXi Su Qi Yi Lan Tang Hai-Lin Liao Ai-Jun Xie Na Liu Liang

【机构】 南华大学肿瘤研究所； 南华大学附属第一医院；

【摘要】 目的:运用血清蛋白质组分析方法,建立人胃癌组织蛋白质双向凝胶电泳图谱以及分别与胃癌患者血清和非癌人群血清的Western blot反应图谱,初步筛选人胃癌相关抗原。方法:运用固相pH梯度(Immobilized pH gradient, IPG)双向凝胶电泳(Two-dimensional electrophoresis,2- DE)方法,分离人胃癌组织的总蛋白质;同一个样品跑三块凝胶,一块考马斯亮染显色作为平行胶,另二块分别以胃癌患者自身血清和非癌人群血清作一抗进行Western blot分析, 获得Western blot反应图谱;用人工与计算机相结合的方法对图像进行比较分析,识别差异反应的蛋白质点,然后与平行胶比较,找出匹配的差异反应蛋白质点;对差异反应的蛋白质点应用基质辅助激光解析电离飞行时间质谱(MALDI- TOF-MS)进行质谱分析鉴定,获得相应的肽质指纹图谱 (peptide mass fingerprint,PMF),数据库搜索鉴定出差异反应的蛋白质。结果:人胃癌组织2-DE后分别与胃癌患者自身血清和非癌人群血清进行免疫印迹,获得分辨率较高的 Western blot反应图谱;图像分析找出差异反应的蛋白质点 14个,质谱鉴定13个蛋白质,分别为:核内不均一核糖核蛋白K(heterogeneous nuclear ribonucleoprotein K,hnRNP K)、核内不均一核糖核蛋白H1(hnRNP H1)、核内不均一核糖核蛋白H2(hnRNP H2)、细胞骨架蛋白18(cytokeratin 18,CK18)、Fkbp52热休克蛋白90复合物(crystal struc- ture of Fkbp52 c-terminal domain complex with the c- terminal peptide meevd of Hsp90)、血清维生素D结合蛋白前体(serum vitamin D-binding protein precursor, DBP)、硒结合蛋白1(selenium binding protein 1,SBP1)、真核细胞翻译延长因子1(eukaryotic translation elonga- tion factor 1 delta,eEF-1)、ACTB蛋白(ACTB protein)、脑肌酸激酶(brain creatine kinase,CKB)、26S蛋白酶体 ATP酶亚单位3(proteasome 26S ATPase subunit 3, PSMC3)、二甲基精氨酸二甲胺水解酶1(dimethylarginine dimethylaminohydrolase 1,DDAH—1)、NADH脱氢酶硫- 铁蛋白1(NADH dehydrogenase Fe-S protein 1)。结论:获得分辨率较高的人胃癌组织总蛋白2-DE图谱;获得分辨率较高的2-DE人胃癌组织蛋白质组与胃癌患者自身血清及非癌人群血清反应的Western blot反应图谱;初步筛选了13个人胃癌相关抗原。为进一步筛选可应用于临床早期诊断、治疗、预后评估及疾病检测的人胃癌分子标志物奠定了基础。更多还原

【Abstract】 Objective: To seek the human gastric carcinoma biomarkers or tumor associated antigens, the western-blot imaging films reacted with the patient autologous serum and with control serum were compared and human gastric carcinoma associated antigens were identified by using Serologic Proteome Analysis (SERPA). Methods: In this study, Serologic Proteome Analysis (SERPA) of ten human gastric carcinoma tissues were performed. Three gels were run simultaneously from the same gastric carcinoma by IPG -2D PAGE. After separation, the proteins of the gel (replica gel) was visualized by Coomassie brilliant blue staining technique. The other two gels were used for western blot analysis. Proteins are transferred to PVDF membranes, and then reacted with autologous patient serum and with control normal serum, respectively. The Western blot imaging films were obtained and the differential reacting protein spots were recognized, then the differential reacting protein spots in the replica gel by matching analysis using artificial method and computer analysis. Finally identified by pep-tide mass fingerprint(PMF) based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Results: The results showed that well-resolved 2-DE western blot imaging films of human gastric carcinoma reacted with autologous patient sera and with control sera were obtained. Fourteen differentially expressed proteins which only were reactive with gastric carcinoma patient serum were found and thirteen proteins, which are human gastric carcinoma associated antigens, were identified by MS. They are heterogeneous nuclear ribonucleoprotein K (hnRNP K), hnRNP H1, hnRNP H2, cytokeratin 18(CK18), crystal structure of Fkbp52 c-terminal domain complex with the c-terminal peptide meevd of Hsp90, serum vitamin D-binding protein precursor(DBP), selenium binding protein 1(SBP1), eukaryotic translation elongation factor 1 delta(eEF-1), ACTB protein, brain creatine kinase(CKB), proteasome 26S ATPase subunit 3(PSMC3), dimethylarginine dimethylaminohydrolase l(DDAH-1), NADH dehydrogenase Fe-S protein 1. Results: By using Serologic Proteome Analysis (SERPA), well-resolved, reproducible 2-DE maps of human gastric carcinoma tissues and 2-DE western blot imaging films of human gastric carcinoma reacted with autologous patient sera and with control sera were obtained in this investigation. Thirteen human gastric carcinoma associated antigens were characterized. These results will provide scenticfic foundation for screening the molecular biomarker used to diagnose, therapy and prevention of human gastric carcinoma, as well as to elevate the patient’ s prognosis and provide new clue for the research of the mechanism of occurrence and development of human gastric carcinoma.更多还原