CAJ | 학술논문

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(voltage-dependent potassium channel 1.3,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5 d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(transcription-polymerase chain raction,RT-PCR)及Western blot,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30 mg/L OxLDL于37℃孵育60 h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.0.1 nmol/L和10 nmol/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＜O.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.
목적:연구인단핵세포원성거서세포향포말세포분화과정중,전압의뢰성갑통도1.3(voltage-dependent potassium channel 1.3,Kv1.3)mRNA화단백적표체급작용.방법:채용밀도제도리심가첩벽점부법,종남성건강지원자적외주혈중분리단핵세포,경5 d배양후분화위거서세포.재건립인거서세포원성포말세포모형적기출상,채용면역세포화학염색법、역전록취합매련반응(transcription-polymerase chain raction,RT-PCR)급Western blot,연구Kv1.3통도적표체,병관찰기특이성조단제--rMargatoxin대섭취양화수식저밀도지단백(OxLDL)적거서세포담고순대사적영향.결과:장거서세포동30 mg/L OxLDL우37℃부육60 h후,세포체적증대,병유허다홍색적지질과립침적우세포질내,세포내적총담고순(TC)、유리담고순(FC)급담고순지(CE)적함량균현저증가,CE/TC종(14.4±6.8)%제고도(57.9±3.5)%(n=7,P＜0.05);이Kv1.3통도mRNA급단백적표체수평몰유명현개변.0.1 nmol/L화10 nmol/L적rMargatoxin능현저감소거서세포내TC、FC급CE적함량,CE/TC분별강지(42.8±11.6)%화(22.6±8.0)%(n=7,P＜O.05),세포내침적적홍색지질과립야명현감소.결론:인단핵세포원성거서세포향포말세포분화과정중,Kv1.3통도적표체무명현개변.특이성조단Kv1.3능방지포말세포형성.
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. r Margatoxin (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 Ky1.3 channel does not change obviously during human monocyte-derived macrophage differentiation into foam cells and the blocking of it would prevent foam cell formation.