Investigation of hrDNA targeting vector-mediated tumor-specific suicide gene therapy for hepatocellular carcinoma

查看全文 作　　者：WANG Lina XUE Zhigang LI Zhuo XUE Jinfeng LIU Xionghao PAN Qian LONG Zhigao CAI Fang WU Lingqian DAI Heping XIA Kun LIANG Desheng XIA [1]Jiahui 机构地区：[1]National Lab of Medical Genetics of China, Central South University Changsha 410078, China 出　　处：《Chinese Science Bulletin》索引2006年第51卷第19期,共9页 基　　金：This work was supported by the National Natural Science Foundation of China(Grant No.30500302);Chinese 973 Projects(Grant No.2004CB518800);863 Projects(Grant No.2002AA227012). 摘　　要：Human ribosomal DNA (hrDNA) target- ing vector (pHrn) is one of the human derived vectors, which was devised by our lab and has got patent authority. To investigate its effect on gene therapy for hepatocellular carcinoma, a double suicide fusion gene expression cassette, CDUPRT/GFP controlled by a synthetic CMV enhancer-enhanced hTERT promoter (CeTp) which was determined by luciferase assays was constructed in pHrn backbone, creating an expression vector pHr-CeTpCDUPRT/GFP. After transfer of plasmid to hepatocellular carcinoma cell line Bel7402 in vitro, the transfection efficiency reached 30%―50% by using flow cytometer. The expression of CDUPRT/GFP was detected by RT-PCR and Western Blotting. After the administra- tion of 5-FC, high performance liquid chromatography (HPLC) was applied to examine the level of 5-FU in supernatant, resulting in a concentration of 60.15 μg/mL. The Methylthiazolyl tetrazolium (MTT) assay was then utilized to investigate the antitumor effect of pHr-CeTpCDUPRT/GFP in Bel7402 cells, and rela- tive cell survival of 60%-35% was observed after 5-FC treatment. In vivo experiments, the nude mouse model of hepatocellular carcinoma was constructed and in situ gene therapy was performed. The results indicated the tumor growth of treatment group was obviously suppressed, and some even shrank, whenthe vectors and prodrugs were injected continuously. The expression of CDUPRT in tumor tissues was also identified by RT-PCR, and the concentration of 5-FU was 7.694 μg/mL in blood serum using HPLC detection. Then the pathological section of tumor tissues revealed significant tumor cell necrosis. All of these results provide important experiment evidences of the gene therapy for hepatocellular carcinoma with the utilization of our vectors. 关 键 词：强化因子 hrDNA HTERT启动子 自杀基因 肝细胞癌 基因治疗