【摘要】 目的:采用抗人血管内皮生长因子(vascular endothelial growth factor,VEGF)发卡状核酶基因转染肝癌细胞,观察核酶对VEGF表达及肿瘤生长的影响。方法:采用脂质体介导的方法,将人工合成的抗VEGF发卡状核酶基因转染肝癌细胞SMMC-7721,同时制备空载体和细胞对照,经G418筛选获得阳性克隆,基因组水平鉴定核酶在细胞中的表达,半定量RT-PCR法和免疫组化法观察核酶对SMMC-7721细胞VEGF表达的影响。接种各组细胞于裸鼠皮下,观察瘤体体积大小和质量,免疫组化法观测各组肿瘤微血管变化和VEGF的表达。结果:核酶基因成功导入到肿瘤细胞中,转基因细胞的增殖速率明显下降(P<0.01),转染核酶组细胞VEGF水平明显下降(P<0.01)。移植瘤成瘤率和生长速度减慢(P<0.01),肿瘤组织VEGF表达和血管形成减少(P<0.01)。结论:抗人VEGF发卡状核酶基因在体内、体外均可抑制肝癌VEGF表达,通过减少血管形成抑制肿瘤生长,为进一步开展肝癌血管靶向基因治疗提供实验依据。更多还原

【Abstract】 Objective:Hepatocarcinoma cell line was transfected with anti-vascular endothelial growth factor(VEGF) hairpin ribozyme gene to observe the effect of hairpin ribozyme on VEGF expression and the growth of the xenografted tumors.Methods:The artificial anti-VEGF hairpin ribozyme gene was transfected into hepatocarcinoma SMMC-7721 cells via lipofectin mediation.The blank vector and the cell controls were prepared simultaneously. Then,positive clones were screened by genticin(G418).The transcription of ribozyme was confirmed by RT-PCR.The effects of the ribozyme on VEGF expression of SMMC-7721cells were detected by semi-quantitative RT-PCR and immunohistochemical method.Cells in each group were inoculated into nude mice.The tumor volume and weight were recorded.The change in microvessel density and expression of VEGF was determined by immunohistochemistry.Results:Ribozyme gene was successfully transferred into tumor cells.The proliferation rate of ribozyme-transfected SMMC-7721 cells was significantly slower(P<0.01).The expression of VEGF significantly decreased in ribozyme-transfected SMMC-7721 cells.After rebozyme transfection,the tumor formation rate significantly decreased and the growth speed of xenografted hepatocarcinoma markedly slowed down.The microvessel density and angiogenesis of the xenografted hepatocarcinoma were obviously reduced.Conclusion:Anti-VEGF hairpin ribozyme gene significantly inhibited the VEGF expression of hepatocarcinoma in vitro and in vivo by inhibiting angiogenesis of tumor cells.This study provided an experimental evidence for anti-angiogenesis gene therapy for hepatocarcinoma.更多还原