核糖体基因区打靶载体pHrn是本室构建的人源基因载体之一，为探讨其在肝癌基因治疗的作用，本研究构建了pHr—CeTpCDUPRT／GFP质粒载体并进行了肝癌的体内外治疗实验研究．该质粒载体以pHrn载体为骨架，CMV＋hTERT启动子作为转录调控元件，CDUPRT／GFP自杀融合基因为目的基因．体外转染肝癌细胞BEL7402后，流式细胞仪检测转染效率为30％～50％；RT—PCR和Western blotting结果表明，有CDUPRT表达；HPLC检测5-FC可转化为5-Fu，给药后12 h 5-Fu浓度达60．15μg／mL；四唑盐（Methylthiazolyl tetrazolium，MTT）分析结果显示，200～800μg／mL5-FC使BEL7402的平均存活率为60％～35％．同时，对裸鼠肝癌模型进行瘤内转染，结果显示，当持续注射质粒和前体药物治疗时，裸鼠肿瘤生长明显被抑制，甚至体积稍有缩小；RT—PCR检测结果表明，瘤内有CDUPRT表达；HPLC检测裸鼠血清中的5-FU浓度为7．694μg／mL；肿瘤组织病理切片显示，大量肿瘤细胞坏死．这些结果为实际应用此载体进行肝癌基因治疗提供了重要的实验依据．
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.
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 Jiahui
