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目的分研究双氯芬酸对人巨噬细胞电压依赖性钾通道Kv1.3、内向整流钾通道Kir2.1表达的影响及意义。方法以健康人外周血单核细胞源性巨噬细胞为对象,采用Real-time RT-PCR及Western blot技术研究双氯芬酸对Kv1.3和Kir2.1表达的影响;电压敏感染料膜电位标测技术分析膜电位的变化,并用酶荧光化学法检测摄取氧化修饰低密度脂蛋白(OxLDL)的巨噬细胞内胆固醇酯(CE)的构成比率。结果双氯芬酸(1.5和15μmol/L)抑制巨噬细胞Kv1.3和Kir2.1的表达。同对照组相比,Kv1.3mRNA下降分别超过80%和90%(P<0.05),Kir2.1 mRNA下降分别超过20%和30%(P>0.05);两种钾通道蛋白水平的下降均分别超过10%和60%,且存在明显的剂量依赖性(P<0.05)。同时,双氯芬酸可剂量依赖性减弱巨噬细胞表面的荧光强度,使膜电位分别下降约28%和54%(P<0.05)。巨噬细胞同30 mg/L OxLDL孵育60 h后,细胞体积明显增大,且有许多红色的脂质颗粒沉积于细胞质内,CE/TC的百分比超过50%。1.5和15μmol/L双氯芬酸分别使摄取OxLDL的巨噬细胞内CE的百分比显著减少到(23.624±3.34)%和(13.601±2.916)%,但缺乏明显的量效关系(P>0.05)。结论双氯芬酸显著下调人巨噬细胞Kv1.3和Kir2.1的表达,降低细胞膜电位,并抑制泡沫细胞形成。
Objective To investigate the effect and significance of diclofenac on the expression of Kv1.3, an inward rectifier potassium channel, and Kir2.1 in human macrophages. Methods The effects of diclofenac on the expression of Kv1.3 and Kir2.1 were studied by Real-time RT-PCR and Western blot in healthy human peripheral blood monocyte-derived macrophages. The voltage-sensitive dye membrane potential measurement The change in membrane potential was analyzed and the constituent ratio of macrophage cholesterol ester (CE) uptake of oxidized low density lipoprotein (OxLDL) was measured by enzyme fluorescence spectroscopy. Results Diclofenac (1.5 and 15 μmol / L) inhibited the expression of Kv1.3 and Kir2.1 in macrophages. Compared with the control group, Kv1.3 mRNA decreased more than 80% and 90% respectively (P <0.05), Kir2.1 mRNA decreased more than 20% and 30% respectively (P> 0.05) Over 10% and 60%, respectively, with significant dose-dependent (P <0.05). Meanwhile, diclofenac attenuated the fluorescence intensity of macrophages in a dose-dependent manner and decreased the membrane potential by about 28% and 54%, respectively (P <0.05). Macrophages incubated with 30 mg / L OxLDL for 60 h, the cell volume increased significantly, and there are many red lipid particles deposited in the cytoplasm, the percentage of CE / TC more than 50%. Diclofenac 1.5 and 15 μmol / L decreased the percentage of CEs in macrophages up to (23.624 ± 3.34)% and (13.601 ± 2.916)%, respectively, but there was no significant dose-response relationship (P> 0.05). Conclusion Diclofenac significantly down-regulated the expression of Kv1.3 and Kir2.1 in human macrophages, decreased the cell membrane potential and inhibited the formation of foam cells.