应用RNA干扰技术体外抑制脂蛋白脂肪酶基因的表达  

In Vitro Inhibition of Lipoprotein Lipase Gene Expression by RNA Interference

作　　者：刘明[1] 赵铁强[1] 冼勋德[1] 高松[1] 范江霖[2] 黄薇[1] 刘国庆[1] 

机构地区：[1]北京大学心血管研究所分子心血管学教育部重点实验室,北京市100083 [2]日本筑波大学医学院病理系心血管病研究室,日本筑波3050031

出　　处：《中国动脉硬化杂志》2005年第1期45-47,共3页Chinese Journal of Arteriosclerosis

摘　　要：目的　给探讨脂蛋白脂肪酶基因作为动脉粥样硬化基因治疗靶点提供实验基础。方法　利用RNA干扰技术在细胞水平抑制脂蛋白脂肪酶基因的表达 ,根据人脂蛋白脂肪酶基因设计靶序列短发夹RNA ,构建重组干扰质粒pSIREN DNR shRNA ,将干扰质粒与人脂蛋白脂肪酶真核表达质粒 pCDNA3 hLPL以脂质体方法共转染COS 7细胞 ,通过实时荧光定量聚合酶链反应、Westernblot和活性检测等方法判断脂蛋白脂肪酶基因抑制的效果。结果　转染干扰质粒的细胞中脂蛋白脂肪酶mRNA、蛋白表达以及酶活性均明显降低 ,抑制率可达 70 %以上。结论　所选靶序列可以有效抑制细胞中脂蛋白脂肪酶基因的表达 。Aim To evaluate the potential inhibition of lipoprotein lipase (LPL), the rate limiting enzyme in hydrolyzing plasma triglyceride, in cultured cells by RNA interference technique, which would be further explored as a new way for intervention of atherosclerosis. Methods Two target sequences were designed according to human LPL sequence information and cloned into a plasmid expressing desirable small hairpin RNA (shRNA) in mammalian cells. This plasmid was co-transfected with LPL expressing plasmid into COS-7 cells. Real time polymerase chain reaction (PCR), Western blot and enzyme activity assay were carried out to determine the inhibition of LPL gene expression. Results Compared with the control cells, more than 70% inhibition of LPL gene expression in cells co-transfected with shRNA was observed at the levels of mRNA, protein and enzymatic activity. Conclusion Expression of LPL gene can be efficiently inhibited in vitro by RNAi technique. These results are informative for further study in vivo.

关 键 词：病理学与病理生理学 脂蛋白脂肪酶基因的RNA干扰 RNA干扰 脂蛋白脂肪酶 短发夹RNA 基因表达 动脉粥样硬化 

分 类 号：R363[医药卫生—病理学]