靶向胆囊癌血管内皮生长因子基因短发夹样RNA表达质粒的构建与筛选

背景:已有研究通过RNA干扰技术,抑制结肠癌、前列腺癌和视网膜母细胞瘤细胞的血管内皮细胞生长因子基因的表达.尚未见关于RNA干扰血管内皮生长因子抑制胆囊癌肿瘤的相关研究.目的:创新性构建编码胆囊癌血管内皮生长因子基因的短发夹样RNA质粒表达载体,筛选出沉默血管内皮生长因子基因效果最明显的短发夹样RNA表达质粒.设计、时间及地点:基因工程观察实验,于2008/2009在中南大学湘雅医院卫生部肝胆肠外科研究中心实验室完成.材料:人胆囊癌细胞株GBC-SD购于同济大学肿瘤研究所.方法:设计4对针对血管内皮生长因子基因不同位点的短发夹样RNA片段.构建携带此短发夹样RNA片段的表达质粒(短发夹样RNA1-4)并行酶切鉴定分析.然后通过脂质体将重组质粒转染到胆囊癌细胞株GBC-SD中,48 h后测定转染率.主要观察指标:重组质粒酶切鉴定结果.转染效率.采用及荧光PCR检测血管内皮生长因子mRNA表达.结果:成功构建了靶向血管内皮生长因子基因的短发夹样RNA质粒表达载体,其中抑制效果最为明显的短发夹样RNA质粒表达载体为pDC316-EGFP-U6-shRNA2 质粒.重组质粒经酶切鉴定证实构建成功.质粒在GBC-SD细胞中的转染率约为58.6%.短发夹样RNA抑制血管内皮生长因子基因的mRNA表达,短发夹样RNA2抑制率最高,可达86%.结论:成功构建并筛选出的靶向血管内皮生长因子的短发夹样RNA表达质粒,其对胆囊癌GBC-SD细胞内的血管内皮生长因子表达具有明显抑制作用.短发夹样RNA2表达质粒抑制作用最明显.

Construction and screening of plasmid expression vectors containing short hairpin RNA targeting at vascular endothelial growth factor gene of GBC-SD cells

BACKGROUND: Previous reseamh has proved that RNA interference can inhibit vascular endothelial growth factor (VEGF) gene expression of colon carcinoma, carcinoma of prostate, and retinoblastoma. However, RNA interference inhibiting VEGF of carcinoma of gallbladder was not reported. OBJECTIVE: To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting hVEGF165 mRNA. DESIGN, TIME AND SETTING: A gene engineering study was performed at National Hepatobiliary & Enteric Surgery Research Center, Xiangya Hospital, Central South University from 2008 to 2009.MATERIALS: Human GBC-SD was provided by Tumor Research Institute of Tongji University. METHODS: Four pairs of shRNAs that targeted at VEGF gene were designed. The eukaryotic expression plasmids (named shRNA1-4) were constructed and identified using restriction enzyme analysis. The plasmids were then transfected into GBC-SD cells via liposome2000. The transfection rate of recombinant plasmids was measured at 48 hours after transfection. MAIN OUTCOME MEASURES: Enzyme analysis of recombinant plasmid; transfection rate; VEGF mRNA expression determined using fluorescent polymerase chain reaction. RESULTS: shRNA plasmid vector targeting at VEGF gene was successfully constructed, in particular, pDC316-EGFP-U6-shRNA2 was the most effective. The expression plasmids were confirmed by restriction enzyme analysis. The transfection rate of recombinant plasmids in GBC-SD cells was approximately 58.6%. shRNA could inhibit VEGF mRNA expression, in particular, the inhibitory rate of RNA2 was the highest by 86%.CONCLUSION: The shRNA eukaryotic expression plasmid targeting at VEGF gene is constructed and selected successfully, and it can remarkably inhibit VEGF expression of GBC-SD cells. Additionally, the inhibitory effect of RNA2 is the greatest.