Ox-LDL对PMA诱导的THP-1巨噬细胞自噬的影响

摘要

目的探讨氧化型低密度脂蛋白( Ox-LDL)对PMA诱导的THP-1巨噬细胞自噬的影响。方法PMA诱导THP-1细胞24 h,使其分化为巨噬细胞,再分别用0、10、20、40或80 mg/L的Ox-LDL处理24 h,逆转录聚合酶链反应( RT-PCR)和免疫印迹技术( Western blotting)分别检测Beclin-1以及LC3的mRNA及蛋白表达；细胞免疫荧光检测LC3在细胞内的含量；MDC染色检测自噬囊泡的形成。结果随着Ox-LDL浓度的增加,THP-1源性巨噬细胞Beclin-1 mRNA及蛋白表达显著降低(P<0.05)；LC3 mRNA表达无明显改变(P>0.05),但蛋白水平LC3II/LC3I显著降低(P<0.001)；免疫荧光结果表明随着Ox-LDL浓度的增加,LC3II含量降低,MDC染色结果显示自噬囊泡随着Ox-LDL浓度的增加而减少。结论 Ox-LDL抑制PMA诱导的THP-1巨噬细胞自噬。%Objective To explore the effect of oxidized low density lipoprotein ( Ox-LDL) on autophagy in THP-1-derived macrophages induced by phorbol-12-myristate-13-acetate ( PMA ) . Methods After treatment with PMA for 24 h,THP-1-derived macrophages were treated with 0,10,20,40 or 80 mg/L Ox-LDL for 24 h. Reverse transcription poly-merase chain reaction ( RT-PCR) and Western blotting were used to detect Beclin 1 and LC3 mRNA and protein expres-sion. Cell immunofluorescence was used to detect intracellular LC3II protein concentration. The autofluorescent substance monodansylcadaverine (MDC) was used to detect autophagic vacuoles (AVs). Results After treated with 0,10,20,40 or 80 mg/L Ox-LDL for 24 h,Beclin-1 mRNA and protein expression were obviously decreased(P<0. 05). There was no obvious change in LC3 mRNA expression (P>0. 05). However,the ratio of LC3II/LC3I was decreased with a concentration dependent manner at the level of protein (P<0. 001). Cell immunofluorescence showed that the intracellular LC3II concen-tration was decreased in response to Ox-LDL concentration. MDC staining showed that AVs had negative correlation with Ox-LDL concentration. Conclusion Ox-LDL inhibited PMA induced-THP-1 macrophage autophagy.