以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deantinase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125 I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胧癌EJ细胞的杀伤作用.人工合成辐射敏感性启动子E8,将启动子克隆至质粒pCD2的CD基因上游,构建以E8为启动子、CD基因为目的基因的新质粒,并采用DNA测序法测定E8和CD基因的序列;脂质体Lipofectamine2000介导pE8-CD转染膀胧癌EJ细胞,用[3]I照射(吸收剂量为2舜)后,蛋白质免疫印迹分析(Western blot)测定CD蛋白表达;在转染EJ细胞中分别加人不同剂量125 I勺和5-FC,四哇盐比色法(MTT法)测定各组细胞存活率,并以未经125 I勺照射组、未加5-FC组和5-氟尿啼吮(5-FU)组(阳性对照组)进行对照.DNA测序显示构建的pE8-CD质粒含E8启动子及CD基因序列;Westem blot可检测到CD基因表达;125 I加5-FC组细胞存活率明显低于未经125 I照射组及未加5-FC组,与5-FU组相近.这表明放射性核素与基因治疗联合对肿瘤细胞具有协同杀伤作用.%To investigate the killing effect on EJ human bladder cancer cells by combination of radiation-responsive gene promoter conjoined CD/S-FC system and ionizing radiation with radionuclide 125 I, plasmid vector containing synthetic gene promoter E8 responsive to ionizing radiation(IR)and CD gene in downstream was constructed, analyzed by DNA sequencing, and transiently transfected into EJ bladder cancer cells by liposome-mediated method. Expression of downstream CD gene was detected via ionizing radiation of radionuclide 1311 at 2Gy by Western Blot. The killing effect induced by different doses of 125I and 5-FC in plasmid-transfected EJ cells was investigated by MTT colorimetric assay. DNA sequencing demonstrated that the plasmid vector contained E8 promoter and CD gene sequences , Western Blot detected the protein CD, and MTT colorimetric assay showed that the death rate of plasmid-transfected EJ cells treated with 125I and 5-FC was greater than the control groups without 125I or 5-FC, furthermore, it was approximate to the positive contrast group treated with 5-FU. The results reveal that the combination radionu-clides and gene therapy play collaborative roles in killing tumor cells.
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