反义VEGF基因转染对HEP-2细胞生物学行为的影响

目的观察反义VEGF基因转染对HEP-2细胞生物学行为的影响.方法克隆人VEGF基因,将VEGF-cDNA反向克隆到真核表达载体pcDNA3,构建反义VEGF表达载体pcDNA3-VEGF(-);利用阳离子脂质体载体,稳定转染人喉癌Hep-2细胞,并通过流式细胞仪检测,观察转染细胞细胞周期的改变,在透射电镜下观察转染细胞超微结构的改变;将转染细胞注入裸鼠皮下,观察肿瘤生长情况.结果成功克隆了人VEGF基因,并构建了携带反义VEGF基因的真核表达载体pcDNA3-VEGF(-);将其稳定转染人喉癌Hep-2细胞,获得了稳定表达反义VEGF的细胞系,与正常Hep-2细胞相比,该细胞系细胞周期及超微结构均无明显改变;将转染细胞注入裸鼠皮下,与对照组相比,肿瘤的生长速度明显减缓.结论反义VEGF基因转染可以有效地抑制喉癌的生长.

Influence of human antisense-VEGF gene transfection to Hep-2 cells in vitro and in vivo

Objective: To observe the effect of cationic liposome mediated antisense-VEGF gene transfection on the growth of laryngeal cancer Hep-2 cells in vitro and in vivo. Methods: The VEGF-cDNA gene was cloned by RT-PCR from human laryngeal cancer, its eukaryotic expression vector pcDNA3-VEGF(-) with antisense-VEGF gene was constructed and identified by PCR and double-enzyme digestion. The pcDNA3-VEGF(-) was transfected into laryngeal cancer Hep-2 cell line by using cationic liposome (LP 2000). Then the Hep-2 cells transfected with pcDNA3-VEGF(-) was tested by Flow Cytometer (FCM) and transmission electron microscopy. The Hep-2 cells (transfected with pcDNA3-VEGF(-), pcDNA3 and no-transfection) were injected into nude mice respectively, and the size of tumor from different groups was observed. Results: The human VEGF-cDNA was successfully cloned and its eukaryotic expression vector with antisense-VEGF was constructed. The antisense-VEGF gene was transfected into Hep-2 cell line by using cationic liposome (LP2000). The cell cycline and cell ultrastructure had no change. The size of tumor transfected with pcDNA3-VEGF(-) was significantly smaller than that of control groups. Conclusions: The growth of tumor can be inhibited significantly by antisense-VEGF gene transfection.