发夹结构RNA表达载体特异性抑制结肠癌LoVo细胞绿色荧光蛋白基因表达  

Specific inhibition of GFP expression by small hairpin RNA in colon cancer LoVo cell

作　　者：马晋平[1] 汪建平[1] 詹文华[1] 彭俊生[1] 高劲松[2] 朱孝峰[3] 殷勤伟 

机构地区：[1]中山大学附属第一医院普通外科,广州510080 [2]中山大学达安基因诊断中心 [3]中山大学肿瘤防治中心 [4]Division of Health Science and Technology,Harvard-MIT

出　　处：《中华实验外科杂志》2004年第7期792-794,i001,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目(30271276;30271469);广东省自然科学基金资助项目(021891)

摘　　要：目的构建针对绿色荧光蛋白(GFP)基因的发夹结构RNA表达载体(SHipU6GFP),观察SHipU6GFP对结肠癌LoVo细胞的GFP特异性抑制作用。方法设计合成针对绿色荧光蛋白GFP基因的发夹RNA(shRNA)序列,构建SHipU6GFP质粒。采用报告基因质粒pCXGFP(5510bp)和含有鼠U6启动子pU6(3.3kb)质粒,应用LipofectamineTM2000将pCXGFP质粒和SHipU6GFP质粒转染K562细胞和LoVo细胞。荧光显微镜观察转染效果和不同时间的转染效率。结果质粒pU6和SHipU6GFP经EcoRⅤ和XbaⅠ酶切电泳后,前者出现3.3kb和约300bp条带,而后者出现3.3kb和约350bp条带,与实验设计的针对GFP的shRNA长度相符。说明本实验已成功构建了针对绿色荧光蛋白GFP基因的SHipU6GFP质粒。K562细胞和LoVo细胞中分别观察到转染pCXEGFP质粒和SHipU6GFP质粒前后发绿色荧光的细胞数目明显减少。结论结肠癌LoVo细胞存在RNA干扰现象,可以进行相关RNA干扰抑制实验。Objective To construct a expression vector carrying small hairpin RNA (SHi pU6 GFP) for GFP gene.To study the specific inhibition of GFP gene using SHi pU6 GFP in LoVo cell.Methods The reporter gene plasmid pCX GFP (5 510 bp) and pU6 (3.3 kb) were used in this study.Plasmid pCX GFP,pU6 and SHi pU6 GFP carrying hairpin siRNA for GFP gene were transfected in K562 and LoVo cell by using Lipofectamine TM 2000 according to the manufacturer's instructions.Results The constructed SHi pU6 GFP carrying hairpin RNA for GFP gene and pU6 were proven to be the same as designed by restriction endonuclease analysis.The expression of GFP gene were specifically inhibited by SHi pU6 GFP in LoVo cell.Conclusion There is RNA interference phenomenon in LoVo cell.Short hairpin RNA (shRNA) could induce sequence specific GFP gene silencing in LoVo cell.The specific shRNAs could silence GFP gene.shRNA induced silencing may shed light on the application of RNA interference in colorectal cancer gene therapy.

关 键 词：RNA干扰 绿色荧光蛋白 结直肠肿瘤 SHi-pU6-GFP质粒 

分 类 号：R735.3[医药卫生—肿瘤]