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为研究pcsk9基因沉默后对氧化型低密度脂蛋白(oxLDL)诱导THP-1源性巨噬细胞凋亡的影响,用不同浓度oxLDL处理THP-1源性巨噬细胞48h,Hoechst33258染色检测细胞凋亡,RT-PCR、Western blot分别检测pcsk9 mRNA、NARC-1蛋白的表达.应用Lipofectamine 2000转染3对pcsk9 siRNAs进THP-1源性巨噬细胞中,筛选出最有效的siRNA再转染入THP-1源性巨噬细胞,24h后加入oxLDL处理48h,Hoechst33258染色观察细胞评价细胞凋亡,流式细胞术计数检测细胞凋亡率.结果发现,75mg/LoxLDL处理THP-1源性巨噬细胞48h后,Hoechst33258染色可见大量凋亡细胞.同时RT-PCR、Western blot检测发现,pcsk9 mRNA和NARC-1蛋白质表达量均随oxLDL浓度的增加而增加,75mg/LoxLDL组增加最明显.不同浓度siRNA转染THP-1源性巨噬细胞后,RT-PCR筛选出3对siRNAs的终浓度为80nmol/L均可出现明显的沉默效应.选取此浓度在蛋白质水平检测基因抑制情况,筛选出最有效的一对siRNA.将筛选出来的siRNA转染细胞24h后,再用oxLDL处理48h,Hoechst33258染色及流式细胞计数结果显示,转染siRNA组凋亡明显被抑制.结果表明,在本研究的浓度范围内,随着oxLDL浓度增加pcsk9的表达随之增加,同时,THP-1源性巨噬细胞凋亡也明显增加,75mg/LoxLDL最明显,pcsk9 mRNA和蛋白质的表达也在该浓度最高.提示pcsk9 siRNA能有效抑制pcsk9基因的表达,从而有效抑制由oxLDL诱导的THP-1源性巨噬细胞的凋亡.
To investigate the effect of pcsk9 gene silencing on apoptosis of THP-1-derived macrophages induced by oxLDL (oxLDL), THP-1-derived macrophages were treated with oxLDL for 48h and Hoechst33258 staining The expression of pcsk9 mRNA and NARC-1 protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. Three pairs of pcsk9 siRNAs were transfected into THP-1-derived macrophages by Lipofectamine 2000. The most effective siRNA THP-1-derived macrophages were treated with oxLDL for 24 h, then treated with oxLDL for 48 h, Hoechst33258 staining was used to evaluate cell apoptosis, and flow cytometry was used to detect the apoptosis rate.Results showed that 75 mg / L oxLDL treated THP-1-derived macrophages 48h later, a large number of apoptotic cells were observed by Hoechst33258 staining.At the same time, the expression of pcsk9 mRNA and NARC-1 protein increased with the increase of oxLDL concentration and the most obvious increase in 75mg / LoxLDL group.At the same time, After THP-1-derived macrophages were transfected by siRNA, 3 pairs of siRNAs were screened for the final silencing effect of 80nmol / L.This concentration was used to detect gene inhibition at the protein level, Effective pair of siRNAs The selected siRNA transfected cells 24h, then treated with oxLDL 48h, Hoechst33258 staining and flow cytometry results showed that transfected siRNA group was significantly inhibited apoptosis.The results showed that in the concentration range of this study, with oxLDL At the same time, the apoptosis of THP-1-derived macrophages was also significantly increased, the most obvious 75mg / LoxLDL, the highest expression of pcsk9 mRNA and protein, suggesting that pcsk9 siRNA can effectively inhibit pcsk9 Gene expression, thereby effectively inhibiting oxLDL-induced apoptosis of THP-1-derived macrophages.