稳定高表达UroplakinⅡ基因人膀胱癌细胞株的建立及其意义  

Establishment of human bladder cancer cell line stably expressing Uroplakin II gene and its significance

作　　者：陈方敏[1] 童强松[1] 曾甫清[1] 郑丽端[2] 汪良[1] 董继华[3] 鲁功成[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院泌尿外科,武汉430022 [2]华中科技大学同济医学院附属协和医院病理科,武汉430022 [3]华中科技大学同济医学院附属协和医院中心实验室,武汉430022

出　　处：《中华实验外科杂志》2004年第11期1333-1336,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目 (30 2 0 0 2 84)

摘　　要：目的　建立稳定高表达UroplakinII(UPII)基因的人膀胱癌细胞株。方法　采用分子克隆技术构建含全长UPII结构基因的真核表达载体 ,进行酶切鉴定、核酸序列分析 ,并将其在阳离子脂质体Lipofectamine 2 0 0 0的介导下转染UPII基因缺陷型膀胱移行细胞癌 (TCC)细胞株EJ ,经G418筛选获得抗性亚克隆细胞株 ;逆转录 聚合酶链反应 (RT PCR)、Westernblotting方法对亚克隆细胞株进行UPII基因表达鉴定。结果　所获UPII基因真核表达载体pcDNA3 UPII转染EJ细胞后 ,通过G418持续压力筛选 4周获得亚克隆细胞株EJ/UPII ;RT PCR、Westernblotting均未检测到EJ细胞的UPII基因表达 ,而EJ/UPII细胞中UPIImRNA和蛋白表达水平显著增高 (P <0 .0 1)。结论　通过基因转染方法建立了稳定高表达UPII基因的膀胱TCC细胞株 ,为进一步探讨UPII基因在膀胱TCC生物学行为中的作用及其靶向生物治疗策略奠定了基础。Objective To establish bladder cancer cell line with stable high expression of Uroplakin II (UPII) gene.Methods The eukaryotic expression vector containing full-length UPII gene was constructed by molecular cloning techniques,analyzed by restrictive digestion reaction and nucleic acid sequencing.Under the induction of cation liposome Lipofectamine 2000,the vector was transferred into bladder transitional cell carcinoma (TCC) cell line EJ,which lacking UPII gene expression.After being selected by G 418,the subclone cell line was obtained.Cellular gene expression of UPII was identified by RT-PCR and Western blotting.Results After EJ cells being transfected by constructed eukaryotic expression vector pcDNA3-UPII,the subclone cell line EJ/UPII was obtained by persistent G 418 selection for 4 weeks.RT-PCR and Western blotting revealed that there was no UPII expression in EJ cells,while high mRNA and protein expression of UPII was significantly detected in EJ/UPII cells (P< 0.01).Conclusion The bladder TCC cell line with stable high expression of UPII gene has been successfully established through gene transfection method,which was a basis for further exploring the roles of UPII gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

关 键 词：细胞株 高表达 人膀胱癌细胞 基因 EJ细胞 TCC 真核表达载体 亚克隆 PCDNA3 酶切 

分 类 号：R737.14[医药卫生—肿瘤] R735[医药卫生—临床医学]