【摘要】 目的:研究人单核细胞源性巨噬细胞向泡沫细胞分化过程中,电压依赖性钾通道1.3(voltagedependentpotassiumchannel1.3,Kv1.3)mRNA和蛋白的表达及作用。方法:采用密度梯度离心加贴壁黏附法,从男性健康志愿者的外周血中分离单核细胞,经5d培养后分化为巨噬细胞。在建立人巨噬细胞源性泡沫细胞模型的基础上,采用免疫细胞化学染色法、逆转录聚合酶链反应(transcriptionpolymerasechainraction,RTPCR)及Westernblot,研究Kv1.3通道的表达,并观察其特异性阻断剂———rMargatoxin对摄取氧化修饰低密度脂蛋白(OxLDL)的巨噬细胞胆固醇代谢的影响。结果:将巨噬细胞同30mg/LOxLDL于37℃孵育60h后,细胞体积增大,并有许多红色的脂质颗粒沉积于细胞质内,细胞内的总胆固醇(TC)、游离胆固醇(FC)及胆固醇酯(CE)的含量均显著增加,CE/TC从(14.4±6.8)%提高到(57.9±3.5)%(n=7,P<0.05);而Kv1.3通道mRNA及蛋白的表达水平没有明显改变。0.1nmol/L和10nmol/L的rMargatoxin能显著减少巨噬细胞内TC、FC及CE的含量,CE/TC分别降至(42.8±11.6)%和(22.6±8.0)%(n=7,P<0.05),细胞内沉积的红色脂质颗粒也明显减少。结论:人单核细胞源性巨噬细胞向泡沫细胞分化过程中,Kv1.3通道的表达无明显改变。特异性阻断Kv1.3能防止泡沫细胞形成。更多还原

【Abstract】 Objective: To investigate the expression of voltage-dependent potassium channel 1.3 (Kv1.3)mRNA and protein during human monocyte-derived macrophage differentiation into foam cells and its function in foam cell formation. Methods: Human peripheral blood monocytes were isolated from healthy male volunteers by density gradient centrifugation and then by adherent method. The obtained monocytes were cultured for 5 days to differentiate into macrophages. Based on establishment of the human macrophage-derived foam cell model, the expression of Kv1.3 channel was investigated by immunocytochemical staining, reverse transcription-polymerase chain raction (RT-PCR) and Western blot. Furthermore, the effects of rMargatoxin, a Kv 1.3 channel-specific inhibitor, on cholesterol metabolism in macrophages incepting oxidized low density lipoprotein (OxLDL) were studied. Results: After the macrophages co-incubated with 30 mg/L OxLDL at 37 ℃ for 60 hours, the cellular volume obviously enlarged and many red lipid granules were deposited in cytoplasm. The total amount of cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) in cells markedly increased and the ratio of CE/TC rose from (14.4±6.8)% to (57.9±3.5)% (n=7, P<0.05). However, the expression of Kv1.3 channel had no significant change. rMargatoxin (0.1 nmol/L and 10 nmol/L) markedly reduced the contents of TC, FC and CE in macrophages and the ratios of CE/TC decreased to (42.8±11.6)% and (22.6±8.0)%, respectively (n=7, P<0.05). Meanwhile, the red lipid granules deposited in the cytoplasm of macrophages also decreased. Conclusion: These data clearly show that the expression of Kv1.3 channel does not change obviously during human monocyte-derived macrophage differentiation into foam cells and the blocking of it would prevent foam cell formation.更多还原