PTEN多克隆抗体制备和原发性肝癌组织PTEN蛋白表达的研究  

Preparation of Anti-PTEN Polyclonal Antibody and Its Expresion in Hepatocellular Carcinoma Tissues

作　　者：万兴旺[1] 王红阳[1] 江明[1] 何雅琴[1] 曹慧芳[1] 刘淑琴[1] 唐亮[1] 邱秀华[1] 吴孟超[1] 

机构地区：[1]第二军医大学东方肝胆外科研究所国际合作信号转导研究中心,上海200438

出　　处：《肿瘤防治杂志》2003年第6期565-568,共4页China Journal of Cancer Prevention and Treatment

基　　金：国家重点基础研究项目 (G19980 5 12 10和 0 1CB5 10 2 0 5 );国家自然科学基金 ( 3 0 0 0 0 15 9;3 982 5 114和 3 983 0 0 80 )

摘　　要：目的 :制备并鉴定抗PTEN(phosphataseandtensinhomologdeletedonchromosome 10 )兔多克隆抗体 ,利用该抗体研究PTEN蛋白在原发性肝癌中表达的临床病理意义。方法 :以PCR扩增合成人PTEN全长cDNA序列 ;与 6组氨酸融合表达载体pPROEXHTb构建原核表达重组体 ,转染进大肠杆菌诱导融合蛋白表达后将其纯化 ;用融合蛋白免疫新西兰大白兔制备抗PTEN多克隆抗体 ,经Western杂交鉴定后用于免疫组织化学检测 6 0例肝癌组织中PTEN蛋白的表达。结果 :采用制备的兔多克隆抗体进行Westernblot的结果显示 ,在瞬时转染了pcDNA3 0 PTEN的MCF 7细胞裂解液中检测 1条相对分子质量约为 4 8× 10 3的特异性蛋白带 ,而转染了空载体pcDNA3 0的MCF 7细胞中未检测到该蛋白条带。免疫组化显示PTEN蛋白定位于肝细胞的胞质内 ,原发性肝癌组织内PTEN蛋白表达阳性率为4 8 33% ,显著低于对应的癌旁肝组织内 10 0 %的检出阳性率。PTEN蛋白的表达水平与肝癌患者的病理分级和有无癌栓显著相关。结论 :获得了作用特异的PTEN兔多克隆抗体 ,该抗体可应用于研究PTEN在肝癌组织中的表达分析。Objectives To prepare rabbit anti PTEN (phosphatase and tensin homolog deleted on chromosome 10) polyclonal antibody and to explore the clinicopathological significance of PTEN protein expression in human primary hepatocellular cacinoma (HCC).Methods PTEN full length cDNA fragment was amplified by PCR and cloned into pPROEX HTb expression vector.(His) 6 tagged PTEN fusion protein was induced by 0 6 mmol/L IPTG and purified by affinity chromatogram.Specific polyclonal antibody against (His) 6 PTEN fusion protein was prepared by immunizing New Zealand rabbit with (His) 6 PTEN fusion protein.PTEN antibody was characterized by Western blot.PTEN protein expression was detected by microwave antigen retrieval immunohistochemistry.Results A specific protein band of 48×10 3 was detected in the lysates of MCF 7 cells transiently transfected with pcDNA3 0 PTEN.PTEN protein was immunohistochemically stained in the cytoplasmic region of hepatocytes.Among 60 patiens with primary hepatocellular carcinoma,only 48 33%(29/60) patients were positive for PTEN protein in cancerous tissues,while 100%(60/60) patients showed PTEN positive in para carcinoma liver tissues.Conclusions A specific polyclonal antibody against PTEN was prepared.The antibody can be used to analyze PTEN protein expression in HCC tissues.Down regulation of PTEN protein expression may be a potential adjuvant parameter of predicting the progression and prognosis of HCC.

关 键 词：肝肿瘤 PTEN蛋白 抗体 免疫组织化学 预后 

分 类 号：R735.7[医药卫生—肿瘤]