重组小鼠血管抑素基因在胆囊癌细胞中的表达及对血管内皮细胞增殖的抑制作用

目的　研究重组小鼠血管抑素基因真核表达质粒转染的胆囊癌细胞能否表达具有抑制血管内皮细胞生长活性的血管抑素蛋白。方法　应用脂质体LipofectAMINE 2 0 0 0将重组小鼠血管抑素基因的真核表达质粒 pcDNA3.1(+) angiostatin转染胆囊癌细胞株GBC SD ,G4 18抗性筛选 ;以Westernblot检测血管抑素的表达 ,以血管内皮细胞增殖分析检测其活性。结果　经Westernblot检测 ,得到高表达血管抑素的胆囊癌细胞克隆 ,其培养上清能有效抑制bFGF刺激的血管内皮细胞增殖 (P <0 .0 5 )。结论　小鼠血管抑素基因真核表达质粒 pcDNA3.1(+) angiostatin转导的胆囊癌细胞能够表达具有生物学活性的血管抑素蛋白 ,在胆囊癌的血管抑制基因治疗中有潜在临床应用价值

Expression of eukaryotic expression plasmid with mouse angiostatin cDNA in gallbladder cancer cells and antiangiogenic activity of angiostatin protein

Objective To investigate the expression of eukaryotic expression plasmid with mouse angiostatin cDNA in gallbladder cancer cells and antiangiogenic activity of angiostatin protein. Methods The recombinant vector pcDNA3.1(+)-angiostatin was transfected into gallbladder cancer cells GBC-SD with liposome Lipofect AMINE 2000. The expression of angiostatin protein was examined by Western blot in the stable cell lines by G418 and its inhibitory effect on the vascular endothelial cells ECV-304 was observed in vitro. Results After 30d of transfection and selection with G418, macroscopic resistant cell clones were formed.Western blot test showed that angiostatin protein was expressed and secreted by GBC-SD cells transfected with pcDNA3.1(+)-angiostatin.The angiostatin protein could inhibit proliferation of vascular endothelial cells ECV-304 dependent on bFGF (P<0.05). Conclusion Gene of angiostatin in pcDNA3.1(+)-angiostatin, which has potential value in antiangiogenic gene therapy of carcinoma, is expressed by transfected gallbladder cancer cells and the angiostatin protein has antiangiogenic activity.