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AIM:To explore a safe and efficient strategy of tumortherapy using anti-angiogenetic agents.METHODS:Endostatin gene with a signal sequence ofhuman IgG γ chain was amplified by PCR and cloned intopVAX1 plasmid which was the only vector authorized byFDA in clinical trial to construct a recombinant plasmidnamed as pVAX-sEN.The recombinant plasmid wasdetected with EcoRI/KpnI and DNA sequencing.BALB/c micebearing hepatocarcinoma cell line H22 were treated withnaked pVAX-sEN or liposome-DNA complex in which thedose of DNA and the ratio of DNA and liposome weredifferent from each other.To compare the efficiency ofgene transfection,expression of endostatin at the treatedtumor site was assayed with ELISA.To investigate the effectof pVAX1-sEN on hepatocellular carcinoma,pVAX-sENeither naked or in liposome-DNA complex was injectedinto BALB/c mice bearing H22,then the diameter of tumorswas measured,microvessel density was detected byimmunohistochemistry,endostatin expression in vivo wasassayed at different time points.RESULTS:DNA sequencing showed the endostatin genewith the signal peptide was correctly cloned.In situ geneexpression assay indicated that both the ratio of DNAand liposome and the dose of DNA could affect the genetransfection efficiency.Interestingly,naked pVAX-sEN hada similar in situ endostatin expression to pVAX-sEN withliposome.Animal experiments showed that pVAX-sENtogether with pVAX-sEN-liposome complex could efficientlysuppress the growth of mouse hepatoma cells.CONCLUSION:Naked endostatin plasmid intratumoralinjection can get a similar gene transfection efficiency toliposome-DNA complex when used in situ.
AIM: To explore a safe and efficient strategy of tumortherapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgG γ chain was amplified by PCR and cloned intopVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid asd pVAX-sEN.The recombinant plasmid wasdetected with EcoRI / KpnI and DNA sequencing.BALB / c micebearing hepatocarcinoma cell line H22 were treated withnaked pVAX-sEN or liposome-DNA complex in which thedose of DNA and the ratio of DNA and liposome weredifferent from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. Check the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sENeither naked or in liposome-DNA complex was injected in BALB / c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo wasassayed at different time points .RESULTS: DNA sequencing showed the endostatin genewith the signal peptide was correctly cloned.In situ geneexpression assay indicated that both both the ratio of DNA and liposome and the dose of DNA could affect the gene transfer efficiency.Interestingly, naked pVAX-sEN hada similar in situ endostatin expression to pVAX-sEN withliposome.Animal experiments showed that pVAX-sEN-liposome complex could efficientlysuppress the growth of mouse hepatoma cells.CONCLUSION: Naked endostatin plasmid intratumoralinjection can get a similar gene transfection efficiency toliposome-DNA complex when used in situ.