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作 者:李玲[1] 张春丽[1] 李囡 康磊[1] 卢霞[1] 张丽[1] 闫平[1] 王荣福[1]
机构地区:[1]北京大学第一医院核医学科,北京100034 [2]北京市肿瘤防治研究所,北京100083
出 处:《标记免疫分析与临床》2011年第5期329-333,共5页Labeled Immunoassays and Clinical Medicine
基 金:北京市自然科学基金资助项目;项目编号:70831152;北京市自然科学基金资助项目(放射敏感性基因联合放射免疫治疗对膀胱癌的治疗作用与毒性研究);项目编号:7112129
摘 要:以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deaminase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胱癌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转染膀胱癌EJ细胞,用131I照射(吸收剂量为2Gy)后,蛋白质免疫印迹分析(Western blot)测定CD蛋白表达;在转染EJ细胞中分别加入不同剂量125I和5-FC,四唑盐比色法(MTT法)测定各组细胞存活率,并以未经125I照射组、未加5-FC组和5-氟尿嘧啶(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;Western blot可检测到CD基因表达;125I加5-FC组细胞存活率明显低于未经125I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/5-FC system and ionizing radiation with radionuclide ^125I,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 131I 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 radionuclides and gene therapy play collaborative roles in killing tumor cells.
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