小鼠CD154真核表达质粒的构建及其对膀胱癌细胞体内成瘤性的影响  

Construction of recombinant murine CD154 eukaryotic expression plasmid and its effect on the tumorigenicity of bladder transitional cell carcinoma

作　　者：吴建臣 杨宇如[2] 曾浩[2] 夏庆杰[3] 卢一平[2] 管德林 

机构地区：[1]清华大学第一附属医院泌尿中心,北京100016 [2]四川大学华西医院泌尿科 [3]四川大学眼科分子遗传学实验室

出　　处：《中华实验外科杂志》2005年第9期1053-1055,共3页Chinese Journal of Experimental Surgery

摘　　要：目的构建小鼠CD154真核表达质粒,观察其转染膀胱癌细胞后对癌细胞体内成瘤性的影响。方法逆转录聚合酶链反应(RTPCR)法扩增T739小鼠CD154cDNA,重组到pcDNA3.1/Zeo(+)构成重组质粒,经阳离子脂质体转染小鼠膀胱移行细胞癌细胞BTT739,将转染后的细胞接种T739小鼠,观察肿瘤在体内的生长情况。结果RTPCR产物进行凝胶电泳可见0.8kb处的目的基因条带,序列测定结果正确;CD154重组质粒转染BTT739细胞后能够在肿瘤细胞内表达;转染CD154的肿瘤细胞在小鼠体内不能成瘤。结论成功构建小鼠CD154真核表达质粒,将其转染肿瘤细胞后能抑制肿瘤细胞的体内成瘤性。Objectives To construct the recombinant murine CD154 eukaryotic expression plasmid, and investigate its effect on the tumorigenicity of bladder transitional cell carclnoma（TCC）. Methods CD154 cDNA was cloned from the murine splenic lymphocy-tes and inserted into the eukaryotic expression plasmld pcDNA3.1/Zeo（ ＋ ）. The recombinant CD154 plasmid was transfected into BTT739 cells, a kind of TCC of poor differentiation from mouse. Transfected cells were selected with Zeocin for stable expression（BTT739-CD154 and BTT739-pcDNA3.1/Zeo ＋ ）. In vivo tumor growth of the BTT739, BTT739-CD154 and BTT739-peDNA3.1/Zeo（ ＋ ） tumor cells in syngeneic T739 mice was compared. Results Gel electrophoresis, restrictive disgestion and gene sequencing proved the successful construction of CD154 recombinant plasmid. The recombinant plasmid was successfully transfected into cultured BTT739 cells, and its expression was conformed by fluorescence immumohistochemistry. BTT739-CDI54 tumor growth was significantly suppressed in syngeneic mice compared with BTT739 and BTT739-pcDNA3. 1/Zeo （ ＋ ） cells. Conclusion The recombinant murine CD154 eukaryotic expression plasmid was successfully constructed, and its expression on tumor cells can suppress the tumor growth in vivo. Its role in the pathogenesis of CD40 positive tumor and CD154/CD40-based immunotherapy needs luther research.

关 键 词：膀胱肿瘤 基因表达 CD154 质粒 T739小鼠 真核表达质粒 膀胱癌细胞 成瘤性 体内 逆转录-聚合酶链反应 

分 类 号：R737.14[医药卫生—肿瘤]