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目的研究四环素 tet-off 调控系统对 DT_(390)-VEGF_(165)及DT_(390)-VEGF_(exon7)融合基因的调控作用,探索毒素融合基因在调控状态下治疗肝癌的有效途径。方法构建四环素 tet-off 调控的 DT_(390)-VEGF_(165)或 DT_(390)-VEGF_(exon7)融合基因真核表达载体,用脂质体介导转染肝细胞癌 HepG2,在四环素有或无的情况下观察肝癌细胞的形态变化,用质粒直接注射法观察构建物对荷肝癌裸鼠的治疗效果,结果 3μg真核表达构建物转染5×10~5个肝癌细胞/孔(24孔板)96h 后发现,无四环素时转毒素融合基因的细胞存活率为20%,对照细胞存活率为77%;四环素存在时转毒素融合基因的细胞存活率为68%,对照细胞存活率为74%;转染细胞在四环素有或无的情况下经免疫荧光抗体染色均呈现黄绿色荧光,表明融合基因在细胞中得到了表达,且四环素调控系统存在基础量的漏表达;用质粒直接注射瘤体虽可观察到质粒被吸收和表达,但由于表达效率低下,没有明显的治疗效果,结论四环素调控系统基本上可以控制毒素基因在肝癌细胞中的表达。
Objective To study the regulatory effect of the tetracycline tet-off regulatory system on DT_(390)-VEGF_(165) and DT_(390)-VEGF_(exon7) fusion genes, and to explore an effective approach for the treatment of liver cancer with toxin fusion genes under regulatory status. Methods The eukaryotic expression vector of DT_(390)-VEGF_(165) or DT_(390)-VEGF_(exon7) fusion gene regulated by tetracycline tet-off was constructed and transfected into hepatocellular carcinoma HepG2 with liposome. The morphological changes of hepatocellular carcinoma cells were observed under the condition of no or no, and the therapeutic effect of the construct on the hepatic cancer-bearing nude mice was observed by direct plasmid injection method. Results 3 μg of eukaryotic expression construct transfected 5×10-5 hepatoma cells/well (24 After 96 hours, the cell viability of the toxin-fused gene without tetracycline was 20%, the survival rate of the control cells was 77%, and the viability of the transtoxin-fused gene was 68% in the presence of tetracycline, and the viability of the control cells. 74%; Transfected cells showed yellow-green fluorescence with immunofluorescent antibody staining with or without tetracycline, indicating that the fusion gene was expressed in cells, and there was a basal amount of leakage expression in the tetracycline regulatory system; direct injection with plasmids. Although the plasmid could be observed to be absorbed and expressed, it has no obvious therapeutic effect due to low expression efficiency. Conclusion The tetracycline regulatory system can basically control the expression of toxin gene in hepatoma cells.