抗人血管内皮生长因子发夹状核酶基因抑制卵巢癌细胞血管内皮生长因子表达

Inhibition of vascular endothelial growth factor expression of human ovarian cancer cell by anti-VEGF hairpin ribozyme gene.

目的 :研究抗血管内皮生长因子发夹状核酶基因对人卵巢癌细胞血管内皮生长因子 (VEGF)表达的抑制作用 ,探讨卵巢癌基因治疗的新途径。方法 :人卵巢癌细胞SKOV3高表达VEGF ,将抗血管内皮生长因子发夹状核酶基因真核表达载体 (pcDNA3 RZ)转染人卵巢癌细胞SKOV3,经G4 18筛选获得阳性细胞克隆 ,RNA斑点杂交检测核酶基因是否在细胞中表达。采用免疫组化、间接免疫荧光、激光共聚焦显微镜荧光定量法及流式细胞仪结合间接免疫荧光检测转染前后细胞VEGF的表达情况。结果 :转染pcDNA3 RZ组细胞VEGF表达明显降低。结论 :抗人VEGF发夹状核酶基因真核表达载体可有效抑制卵巢癌细胞SKOV3中VEGF的表达 ,有望成为治疗卵巢癌的一种新方法。

Objective:To investigate the possibility whether anti-VEGF hairpin ribozyme gene can be a new therapeutic drug for antiangiogenesis of human ovarian cancer.Methods:There was high expression of VEGF in human ovarian cancer cell line SKOV3.The anti-VEGF hairpin ribozyme gene were transfected into human ovarian cancer cell line SKOV3 using lipofect AMINE.Positive clones were selected by G418 and the expression of ribozyme gene in transfected cell(SKOV3-RZ) was proved by RNA dot blot.The VEGF expressions of SKOV3 cells before or after transfection were detected by immunocytochemical and immunofluorescence methods and laser scanning confocal microscope.Changes in cell surface VEGF protein were determined by flow cytometer immunofluorescence staining analysis as well.Results:VEGF expression was decreased distinctly in SKOV3-RZ cells.Conclusions:Anti-VEGF hairpin ribozyme gene eukaryotic expression vector may inhibit VEGF expression in ovarian cancer cell line and is of significance for ovarian cancer-specific gene therapy.