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作 者:李铁军[1] 康楷[2] 宋建宁[1] 胡瓒斓[1] 何通川[3] 张秉强[4] 张才全[4]
机构地区:[1]重庆市第九人民医院 [2]重庆医科大学附属第二医院 [3]美国芝加哥大学医学中心分子实验室 [4]重庆医科大学附属第一医院,重庆市400016
出 处:《中国肿瘤临床》2007年第7期366-369,373,共5页Chinese Journal of Clinical Oncology
基 金:国家杰出青年基金(编号:30228026);国家青年科学基金资助(编号:30300298)
摘 要:目的:应用RNA干扰技术抑制血管内皮生长因子(VEGF)表达。方法:将VEGF基因作为RNA干扰的靶区,通过E-RNAi网上提供的服务,设计两个特异的RNA干扰序列,将其装入含U6启动子的载体上,构建成抗VEGF基因的小发夹样RNA(shRNA)表达载体,再转染人胚肾细胞HEK293、结肠癌细胞HT29、宫颈癌细胞Hela和肝癌细胞HepG2,通过RT-PCR、Northern杂交和免疫荧光观察VEGF表达受抑的程度。结果:成功构建了两种抗VEGF基因的shRNA表达载体,RT-PCR发现其在HEK293和HT29细胞系中,能明显抑制VEGF基因的表达,抑制率分别为72%和42%,但在Hela和HepG2细胞中的抑制率仅为28%和13%;Northern杂交显示,在HEK293细胞和HT29细胞中,VEGFmRNA明显受抑,抑制率达88%和89%;免疫荧光显示,在HT29细胞中,VEGF蛋白表达的受抑制率为73%。结论:针对VEGF基因的shRNA表达载体能够明显抑制VEGF基因的表达,但在不同细胞系中的作用强度存在差别。Objective: To inhibit expression of Vascular Endothelial Growth Factor (VEGF) by RNA interference and to observe the effect in different cell lines. Methods: The VEGF gene was used as the target area for RNA interference. Two specific RNA series were designed using E-RNAi services and they were put into a vector containing the U6 promoter to construct the short hairpin RNA (shRNA) expression vector. The vector was transfected into human HEK293 embryonic kidney, HT29 colon cancer, Hela cervical carcinoma and Hep G2 hepatocellular carcinoma cells using lipofectamineTM 2000. The level of VEGF mRNA was observed using RT-PCR, Northern-blot hybridization and immunofluorescent staining. Results: Two kinds of VEGF gene-specific shRNA expression vectors were successfully constructed. Effective inhibition of VEGF mRNA expression occurred in the HEK293 and HT29 cells and the inhibition ratio was 72% and 42%, respectively. However, the inhibition ratio was decreased to 28% in the HeLa cells and 13% in HepG2 cells lines. Northern blotting showed that the inhibition rate of VEGF mRNA was 88% and 89% in HEK293 and HT29 cells, respectively. Immunofluorescent staining indicated that the inhibition rate of VEGF protein expression in the HT29 cells was 73%. Conclusion: The expression vector constructed to produce shRNA targeting VEGF can effectively inhibit expression of VEGF, but there are different levels of inhibition in various cells lines.
关 键 词:RNA干扰 血管内皮生长因子 肿瘤细胞 NORTHERN杂交
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