靶向血凝素样氧化型低密度脂蛋白受体1基因的发卡样siRNA表达载体的构建及其效应

A Study in Construction of Short Hairpin Small Interfering RNA Expression Vector Target Human Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Gene and Its Silencing Effect in Human Umbilical Vascular Endothelial Cells

目的设计合成有效的靶向血凝素样氧化型低密度脂蛋白受体1基因的发卡样siRNA(shRNA)表达载体。方法根据siRNA的设计原则,以血凝素样氧化型低密度脂蛋白受体1为靶基因设计并合成小发卡结构两端配对的siRNA寡核苷酸链,再经变性、复性后形成双链血凝素样氧化型低密度脂蛋白受体1shRNA。采用DNA重组技术,将血凝素样氧化型低密度脂蛋白受体1shRNA双链与线性化pGenesil-1质粒表达载体连接,脂质体法转染人脐静脉内皮细胞株,半定量逆转录聚合酶链反应法检测血凝素样氧化型低密度脂蛋白受体1mRNA的表达。结果测序鉴定发现插入的发卡样序列正确,成功合成了发卡样血凝素样氧化型低密度脂蛋白受体1基因RNA干扰表达载体;靶向血凝素样氧化型低密度脂蛋白受体1基因的发卡样siRNA表达载体转染人脐静脉内皮细胞株后,其凝素样氧化型低密度脂蛋白受体1mRNA的表达显著下调。结论成功构建了能有效抑制血凝素样氧化型低密度脂蛋白受体1mRNA表达的发卡样血凝素样氧化型低密度脂蛋白受体1基因RNA干扰表达载体,为进一步利用RNA干扰技术防治动脉粥样硬化提供一种研究基础。

Aim To construct the short hairpin small interfering RNA（shRNA）eukaryotic expression vector specific to human lectin-like oxidized low density lipoprotein receptor-1（LOX-1）gene and to observe its silencing effect on LOX-1 in human umbilical vascular endothelial cells（hUVEC）.Methods The pGenesil-1-LOX-1-shRNA expression vector was constructed by gene recombination,then transfected into the cultured hUVEC.At 48 h after transfection,the stable transfection and expression of LOX-1 mRNA in hUVEC were determined by semi-quantitative RT-PCR. Results pGenesil-1-LDX-1-shRNA expression vector was successfully constructed. DOTAP-mediated gene transfection of pGenesil-1-LOX-1-shRNA expression vector into hUVEC down-regulated the mRNA expression level of LDX-1 gene, as compared with the control group. Conclusion The pGenesil-1-LOX-1-shRNA expression vector can inhibit the expression of LDX-1 mRNA in hUVEC, which may be beneficial in searching new gene therapy of atherosclerosis.