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为了探讨反义VEGF1 65基因对食管癌的抑制作用 ,并初步探讨利用肿瘤低氧微环境改善基因治疗的效果 ,采用PCR技术和DNA重组技术构建了含低氧反应元件的真核表达载体 ,并用此载体构建了含荧光素酶报告基因和反义VEGF1 65基因的重组载体。用脂质体将重组载体导入食管癌细胞 ,体外用化学发光光度计测定低氧对报告基因表达的调节和ELISA法间接测定低氧对反义VEGF基因表达的调节作用。体内利用裸鼠皮下移植实验研究低氧对反义VEGF1 65基因抑瘤作用的影响。体外实验表明 ,用带低氧反应元件的重组真核表达载体转染食管癌细胞 ,在低氧培养下可以使报告基因的表达提高 3 780 % ,并可以显著提高反义VEGF1 65基因的表达 ,体内用带低氧反应元件的载体将反义VEGF1 65基因导入食管癌细胞中 ,其抑瘤效果显著优于不含该元件的载体 ,抑瘤率分别为 71 .7%和 5 6 .1 %。反义VEGF1 65基因能显著抑制食管癌的生长 ;利用肿瘤低氧可以实现治疗基因的自主调节 ,改善基因治疗的效果
In order to investigate the inhibitory effect of antisense VEGF1 65 gene on esophageal cancer and to explore the effect of using gene silencing in tumor hypoxia microenvironment, we constructed an eukaryotic expression vector containing hypoxia response elements by PCR and DNA recombination This vector was constructed with a luciferase reporter gene and antisense VEGF1 65 recombinant vector. The recombinant vector was transfected into esophageal cancer cells by liposomes. The expression of reporter gene was measured by chemiluminescence photometer and the regulation of hypoxia on the expression of antisense VEGF gene by indirect ELISA. Effects of hypoxia on the antitumor activity of antisense VEGF1 65 gene in nude mice by subcutaneous transplantation in vivo. In vitro experiments showed that transfection of esophageal cancer cells with the recombinant eukaryotic expression vector carrying hypoxia response element can increase the expression of reporter gene by 3780% under hypoxic culture and significantly increase the expression of antisense VEGF165 gene, In vivo, the antisense VEGF1 65 gene was transfected into esophageal cancer cells by using the vector with hypoxia response element, and its antitumor effect was significantly better than that of the vector without antisense VEGF1 65 gene. The antitumor rates were 71.7% and 56.1% . Antisense VEGF1 65 gene can significantly inhibit the growth of esophageal cancer; the use of tumor hypoxia can achieve autonomic regulation of therapeutic gene to improve the effect of gene therapy