Vascular endothelial growth factor （VEGF） is an angiogenic regulator that stimulates endothelial cell migration, proliferation, and angiogenesis. VEGF gene therapy is a new promising approach to induce therapeutic a...Vascular endothelial growth factor （VEGF） is an angiogenic regulator that stimulates endothelial cell migration, proliferation, and angiogenesis. VEGF gene therapy is a new promising approach to induce therapeutic angiogenesis for the treatment of ischemic myocardial and limb diseases. Recently, clinical studies have demonstrated successful outcomes using plasmids, retroviruses and adenoviruses.l-3 But the safety of those vectors is poor, which has become a serious concern. Besides immunogenicity caused by viral vectors, a further problem is that viral vectors show a preference to integrate into the transcription or control region of active genes, which may induce tumors and other disorders.展开更多
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 casset...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.展开更多
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 carcin...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.展开更多
基金a grant from the National Natural Science Foundation of China(No.30270734)
文摘Vascular endothelial growth factor （VEGF） is an angiogenic regulator that stimulates endothelial cell migration, proliferation, and angiogenesis. VEGF gene therapy is a new promising approach to induce therapeutic angiogenesis for the treatment of ischemic myocardial and limb diseases. Recently, clinical studies have demonstrated successful outcomes using plasmids, retroviruses and adenoviruses.l-3 But the safety of those vectors is poor, which has become a serious concern. Besides immunogenicity caused by viral vectors, a further problem is that viral vectors show a preference to integrate into the transcription or control region of active genes, which may induce tumors and other disorders.
基金This work was supported by Chinese 973 Projects (Grant No. 2004CB518800)； 863 Projects (Grant Nos. 2002BA7IIA07-08, 2002BA7l1A07-03 & 2002AA227011)；the National Natural Science Foundation of China (Grant No. 31830200) ； Life Science Research Foundation of Hunan Province.
文摘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.
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).
文摘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.
