摘要
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.
In order to study the effect ofpcsk9 siRNA on THP-1 derived macrophages apoptosis induced by oxLDL, THP-1 derived macrophages were induced to differentiate into macrophages by PMA treatment for 24 h. The experiments were designed as follows： cells were incubated with oxLDL with a concentration of 0, 25, 50, 75, 100 mg/L for 48 h respectively. The apoptosis of THP-1 derived macrophages was observed by staining with Hoechst33258. The expression of pcsk9 was analyzed by reverse transcription polymerase chain reaction and Western blot. The siRNAs for pcsk9 gene were designed and synthesized, then transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. RT-PCR and Western blot were conducted to detect the expression ofpcsk9 after 24 h, 48 h respectively. The most efficient siRN. was selected to transfect into THP-1 derived macrophages. 24 h after transfection, cells were treated with oxLDL for 48 h, and then Hoechst 33258 staining. The results showed that the number of cells with nuclear condensation induced by 75 mg/L oxLDL increased significantly. In THP-1 derived macrophages, pcsk9 was upregulated with increasing concentration of oxLDL, while 75 mg/L oxLDL increased significantly. The RNA interference experiment showed that siRNA was successfully transfected into cells and 80 nmol/L as most effective dose of siRNA was selected by RT-PCR and Western blot. Compared with control, the suppression of apoptosis in THP-1 cells transfected with 80 nmol/L of siRNA for 24 h and incubated with 75 mg/L of oxLDL for another 48 h was detected by Hoechst 33258 staining and flow cytometer. Together, these results reveal the expression ofpcsk9 mRNA and protein were increased by oxLDL in a concentration-dependent manner. Expression ofpcsk9 gene could be effectively suppressed by siRNA. The apoptosis of THP-1 derived macrophages induced by oxLDL could be effectively suppressed bypcsk9 siRNA.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2009年第3期323-330,共8页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(30700325)
湖南省科技厅科技计划重点项目(2008FJ2006).~~
