抑制insig1基因表达的siRNA载体的构建和筛选.DOCVIP

[ 基金项目 ] 重庆市卫生局医学科研计划项目(2009-2-208)[ 通讯作者 ] 陈庆伟，电话：（023，E-mail：chenqwcq@Insig-1基因siRNA干扰质粒的构建及对炎症状态下细胞胆固醇代谢的影响柯大智，陈庆伟*，李桂琼，邓玮，莫显刚（400010 重庆, 重庆医科大学: 附属第二医院老年病科)[摘要] 目的　构建针对insig-1 基因的siRNA 表达载体，观察其对炎症状态下Raw264.7小鼠巨噬细胞胆固醇代谢的影响。方法 根据insig-1核苷酸序列, 设计并构建针对insig-1 基因mRNA的3个特异性siRNA表达载体（si-1，si-2，si-3）和1个无同源性的阴性对照载体（si-nc），经酶切和测序鉴定确认 siRNA 载体构建成功后，转染Raw264.7小鼠巨噬细胞， RT-PCR、免疫组化法检测insig-1 基因的表达，筛选出最佳抑制基因后，应用酶学方法和油红O 染色观察炎症处理后Raw264.7细胞内胆固醇(LDL)含量的变化。 结果 经酶切证实，siRNA表达载体构建成功，RT-PCR、免疫组化法显示与未转染组相比，si-1、si-2、si-3转染组Raw264.7细胞中insig-1的mRNA和蛋白表达水平均明显降低(P0.05)，其中si-2干扰效果最强。酶学方法和油红O 染色表明炎症状态下细胞insig-1沉默后胆固醇的含量和油红O 颗粒增多。结论 成功构建了insig-1基因 siRNA干扰质粒，insig1缺失对炎症状态下细胞胆固醇摄取有促进作用。[关键词] insig-1基因；RNA干扰；siRNA质粒载体；巨噬细胞Raw264.7 [中图法分类号 ] R363 [文献标志码 ] AConstruction and identification of siRNA plasmidsfor knockdown of insig-1Ke Dazhi, Chen Qingwei, Li guiqiong, Deng Wei, Mo Xiangang(Department of Geriatrics Cardiology，the Second Affiliated Hospital of Chongqing Medical University Chongqing 400010，China)[ 基金项目 ] 重庆市卫生局医学科研计划项目(2009-2-208)[ 通讯作者 ] 陈庆伟，电话：（023，E-mail：chenqwcq@[Abstract] Objective To construct siRNA plasmids to knock-down insig1, and observe its effecet on cholesterol metabolism in Raw264.7cell after controlled with inflammation． Methods Based on the gene sequence of insig-1, three[ 基金项目 ] 重庆市卫生局医学科研计划项目(2009-2-208)[ 通讯作者 ] 陈庆伟，电话：（023，E-mail：chenqwcq@non-homologous negative control vector (named si-nc) were constructed. After identification by restriction enzyme digestion and sequencing, the vectors were transfected into RAW264.7 cells. The expression of insig-1 was determined by RT-PCR, immunocytochemistry analysis to evaluate the transfection efficiency. The contents of cholesterol in cell were detected by enzymology and Oil Red “O” staining. Results The recombinant vectors were verified by restriction analysis. The expression of insig-1 mRNA and protein was significantly decreased in RAW264.7 cells transfected with si-1, si-2, si-3