Expression of reconstructive hF Ⅷ in the hrDNA by using hrDNA targeting vector

Our lab has constructed a new nonviral vec-tor—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hF Ⅷ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific inte-gration was 2.0×10?5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells?1·24 h?1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.

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

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.

人核糖体DNA打靶载体介导mda-7基因对肝癌的体外基因治疗研究(英文)

人核糖体DNA打靶载体pHr是一种由中南大学医学遗传学国家重点实验室开发构建的针对人类基因组的同源重组质粒载体.利用pHr构建了一种mda-7/GFP融合基因的人源基因表达载体pHr-CMG,并研究了其在肝癌细胞系Bel-7402中的作用.利用荧光显微镜、RT-PCR和Western blotting检测了mda-7/GFP融合基因的表达;利用细胞周期分析、MTT和Hoechst33258染色研究了其在细胞中的作用.结果显示,pHr-CMG载体能在Bel-7402细胞中有效表达MDA-7/GFP融合蛋白,进而抑制细胞增殖和诱导细胞凋亡,推测其可能是由载体表达了mda-7基因引起细胞在G2/M期累积所导致的.同时,实验结果证实了人核糖体DNA打靶载体系统以及pHr-CMG表达载体的有效性,为其在进一步基因治疗研究中的应用提供了理论和实验基础.

核糖体基因区打靶载体电转染肝细胞的孵育条件的优化

目的:提高核糖体基因区打靶载体的转染效率,优化其电转染肝细胞的孵育条件。方法:在其它电转染参数一致的前提下,设计了6组不同的孵育条件,通过分析转染后细胞活率以及48h后目的基因GFP的表达效率,来比较多组不同的孵育条件对电转染效率的影响。结果:6组条件中,转染前21℃,1min,转染后21℃,1min孵育,得到的转染效率最优,比其它的条件得到的转染效率高50%。结论:孵育条件的优化能提高为核糖体基因区靶向表达载体在体外电转染肝细胞的转染效率。