摘要
背景:已有研究发现RNA干扰可通过抑制血管内皮生长因子基因表达抑制肿瘤细胞增殖,但目前关于应用RNA干扰胆囊癌血管内皮生长表达的研究国内外尚无相关报道。目的:构建并筛选靶向血管内皮生长因子的shRNA,观察RNA干扰血管内皮生长因子的效果及对胆囊癌细胞增殖的影响。方法:设计VEGF-shRNA片段,连接到pCYU6/GFP/Neo-shRNA质粒载体上。shRNA转染胆囊癌细胞。建立裸鼠胆囊癌模型,随机分成空白组、阴性对照组、实验组(干扰pShRNA-VEGF2),每组6只。瘤内注射shRNA。荧光显微镜观察细胞生长状态,半定量反转录-聚合酶链反应法与实时荧光定量聚合酶链反应检测RNA干扰效果,观察胆囊癌裸鼠模型肿瘤体积变化。结果与结论:RNA干扰血管内皮生长因子后,胆囊癌细胞细胞变圆,皱缩,部分细胞死亡脱落。shRNA片段血管内皮生长因子2、血管内皮生长因子1均可抑制VEGF mRNA表达,抑制率可达86%,82%。裸鼠肿瘤模型中实验组肿瘤与对照组、阴性组相比明显变小,且生长缓慢(P<0.05),而对照组和阴性组在肿瘤大小及生长趋势上没有明显的差别。实验构建的hVEGF-shRNA质粒表达载体通过抑制血管内皮生长因子的表达,抑制胆囊癌细胞增殖,从而达到间接治疗肿瘤的目的。
BACKGROUND：Previous studies demonstrated that proliferation of cancer cells can be inhibited via RNA interference on the expression of vascular endothelial growth factor （VEGF）. However,few studies report RNA interference on the expression of VEGF in gallbladder carcinoma. OBJECTIVE：To design and screen shRNA targeting VEGF,and to observe the effect of small interfering RNA targeting on proliferation of gallbladder cancer cells. METHODS：The VEGF-shRNA fragment was synthetized and connected with pCYU6/GFP/Neo-shRNA plasmid vector. shRNA was transfected into gallbladder cancer cells. The gallbladder carcinoma models of nude mice were prepared and randomly divided into blank control,negative control and experimental groups,with 6 animals in each group. shRNA was injected into tumor. Cell growth was detected by fluorescence microscope MTT. The RNA interference efficiency was examined by fluorescent quantitative RT-PCR. Changes of tumor volume were also observed. RESULTS AND CONCLUSION：Gallbladder cancer cells were shrunk with round shapes and a part of cells were dead after RNA interference on VEGF. shRNA-VEGF1 and shRNA-VEGF2 could significantly inhibit mRNA gene expression of VEGF,the inhibition ratio was 86% and 82%,respectively. The tumor volume of the experimental group was smaller than the other groups,with slowly growth （P 0.05）. No obvious changes were found in the blank control and negative control groups. The constructed hVEGF-shRNA vector markedly decreases VEGF gene expression and inhibits cellular proliferation,eventually,to treat gallbladder cancer.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第2期258-261,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
中南大学博士研究生学位论文创新项目资助(2009bsxt006)~~
