应用RNA干扰技术体外抑制脂蛋白脂肪酶基因的表达

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、Western blot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,为进一步的体内研究奠定了基础.

In Vitro Inhibition of Lipoprotein Lipase Gene Expression by RNA Interference

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.