转染抗VEGF发夹状核酶基因白血病细胞模型的建立

 目的 建立转染抗血管内皮生长因子（VEGF）发夹状核酶基因的白血病细胞模型，探讨发夹状核酶对白血病细胞系K562 VEGF基因表达的效应。方法 采用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体（pcDNA3-RZ）转染入人白血病细胞系K562，G418抗性筛选获得阳性克隆，转染VEGF pcDNA3-RZ和空载体（pcDNA3）的细胞组均有抗性细胞生长，分别命名为K562-RZ和K562-PC；抽提基因组DNA，用PCR方法验证核酶基因已转染入K562细胞，荧光定量PCR和western blot方法分别检测白血病细胞VEGF mRNA和蛋白的表达量；同时测定K562-RZ，K562-PC和 K562三组培养上清刺激内皮细胞生长的情况。结果 抗VEGF pcDNA3-RZ成功转入白血病细胞系K562，G418筛选2周获得阳性克隆，PCR检测证实核酶基因整合入白血病细胞基因组DNA；与K562及K562-PC细胞相比，转染VEGF核酶基因的K562-RZ细胞VEGF mRNA和蛋白的表达量明显降低；K562-RZ组培养上清对内皮细胞的刺激作用明显弱于K562-PC组。结论 建立了转染VEGF发夹状核酶基因的白血病细胞模型，抗VEGF发夹状核酶基因可明显下调白血病细胞VEGF的表达，为抗肿瘤治疗提供了新的线索。

Establishment of leukemia cell mode transfected with anti-VEGF hairpin ribozyme gene

Objective To establish leukemia cell mode transfected with anti-VEGF hairpin ribozyme gene and explore the possibility of inhibiting expression of VEGF in leukemia cells. Methods The recombinant eukaryotic expression plasmid(pcDNA3-RZ) including anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation and positive clones were screened by G418. Ribozyme gene in K562 cells was conformed by PCR. Fluorescent real time RT-PCR and western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. The growth rate of endothelium cells was detected by MTT assay after leukemia cells serum was added. Results The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418 for 2 weeks. Stable expression of the ribozyme gene in K562 cells was conformed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC(K562 cell transfected with empty vector) cells. The growth of endothelium cells which was added serum of K562-RZ cells was lower than that of control groups. Conclusion The leukemia cell mode transfected with anti-VEGF hairpin ribozyme gene has been established and ribozyme gene can decrease VEGF expression in K562 cells. <