【摘要】 目的:用构建的c-met反义质粒阻断肝细胞生长因子(HGF)/c-met信号传导,探讨c-met信号传导阻滞对卵巢癌细胞血管内皮生长因子(VEGF)的影响。方法:将c-met基因反向克隆入真核表达载体PcDNA3.1中,酶切鉴定结果。用脂质体法将c-met反义基因转染人卵巢癌SKOV3细胞获得阳性克隆,RT-PCR检测转染前后SKOV3细胞中c-met、VEGF mR-NA的表达,蛋白印迹方法检测转染前后SKOV3细胞中VEGF蛋白的变化。结果:获得反向构建的c-met真核表达载体,c-met反义RNA转染卵巢癌细胞后,c-met和VEGF mRNA表达显著降低,VEGF蛋白表达量显著减少。结论:阻断c-met的信号传导可以明显抑制卵巢癌细胞VEGF的分泌,降低肿瘤生长转移的能力,为卵巢癌的基因治疗提供了一定的实验依据。更多还原

【Abstract】 Objective:To construct eukaryotic expressing vector of c-met antisense gene and transfer it into human ovarian cancer cell line SKOV3,and observe the effect of transfected c-met antisense gene on vascular endothelial growth factor(VEGF)of SKOV3 cells.Methods:c-met gene was inserted into eukaryotic expressing vector PcDNA3.1 to construct PcDNA3.1-c-met(-),in which restriction enzyme analysis was used to confirm the vector.The recombinant of PcDNA3.1-c-met antisense plasmid was transfected into human ovarian cancer cell SKOV3 by lipofectamine and positive clone was screened.Reverse transcription polymerase chain reaction(RT-PCR) and Western blot were used to detect the expression of c-met and VEGF at mRNA and protein levels.Results:The PcDNA3.1-c-met vector was obtained.Antisense c-met RNA blocked c-met mRNA expression of ovarian cancer cell SKOV3.VEGF mRNA and protein expression of ovarian cancer cell SKOV3 were significantly decreased after transfected.Conclusion:Inhibition of c-met expression by c-met antisense plasmid can inhibit the expression of VEGF of ovarian cancer SKOV3 in vitro.It is of significance for ovarian cancer specific antisense gene therapy.更多还原