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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细胞内胆固醇含量的变化。结果经酶切证实,siRNA表达载体构建成功,RT-PCR、Western blot、免疫组化法显示与未转染组相比,si-1、si-2、si-3组RAW264.7细胞中insig-1的mRNA和蛋白表达水平均明显降低(P<0.05),其中si-2干扰效果最强。酶学方法和油红O染色结果表明,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,insig-1缺失对炎症状态下细胞胆固醇摄取有促进作用。
Objective To construct the siRNA expression vector targeting the insig-1 gene and observe its effect on the cholesterol metabolism of RAW264.7-induced murine macrophages. Methods Three specific siRNA expression vectors (si-1, si-2, si-3) for insig-1 gene mRNA and one negative control without homology were designed and constructed based on the insig-1 nucleotide sequence. The vector (si-nc) was confirmed by restriction analysis and sequencing. The siRNA vector was successfully constructed and transfected into macrophages of RAW264.7 mice. The expression of insig-1 gene was detected by RT-PCR and immunohistochemistry. After the gene was inhibited, the changes of the cholesterol content in the RAW264.7 cells after inflammation treatment were observed by enzymatic method and oil red O staining. RESULTS: The siRNA expression vector was successfully constructed and confirmed by RT-PCR, Western blot and immunohistochemistry. Compared with the untransfected group, the expression of insig in si-1, si-2 and si-3 groups -1 mRNA and protein expression levels were significantly lower (P <0.05), of which si-2 interference strongest. The results of enzymology and oil red O staining showed that after the insig-1 cells were silenced, the content of cholesterol and the number of oil-red O particles increased after inflammation. Conclusion The siRNA plasmid of insig-1 gene was successfully constructed. The deletion of insig-1 can promote the uptake of cellular cholesterol in the inflammatory state.