外被体蛋白Ⅰ参与载脂蛋白A-1介导泡沫细胞胆固醇流出的作用研究  

Study on the role of coatomer proteinⅠin apolipoprotein A-1-mediated cholesterol efflux from foam cells

作　　者：洪伟涛 张飞龙 黄婕 梁钰敏 陈耿基 陈晓佳 马卫列 丁航 张志珍 HONG Weitao;ZHANG Feilong;HUANG Jie;LIANG Yumin;CHEN Gengji;CHEN Xiaojia;MA Weilie;DING Hang;ZHANG Zhizhen(School of Basic Medical Sciences,Guangdong Medical University,Dongguan,Guangdong 523808,China)

机构地区：[1]广东医科大学基础医学院,广东东莞523808

出　　处：《重庆医学》2025年第3期580-587,共8页Chongqing Medical Journal

摘　　要：目的建立人单核THP-1巨噬细胞源性泡沫细胞模型,分析外被体蛋白Ⅰ(COPⅠ)的α-COP亚基在载脂蛋白A-1(aopA-1)介导泡沫细胞胆固醇流出过程中的作用。方法采用佛波酯(PMA)诱导THP-1细胞贴壁(PMA组)后加入乙酰化低密度脂蛋白(Ac-LDL),建立THP-1巨噬细胞源性泡沫细胞模型(Ac-LDL组),在此基础上加入apoA-1得到apoA-1组细胞。采用液体闪烁计数法和油红O染色检测apoA-1介导的泡沫细胞胆固醇流出率及细胞内的脂质蓄积。通过实时荧光定量PCR(qPCR)、蛋白免疫印迹(Western blot)法和激光共聚焦显微镜观察分析泡沫细胞α-COP的表达情况。用Scrambled shRNA(Scr组)、α-COP shRNA(α-COP shRNA组)慢病毒感染apoA-1介导的THP-1巨噬细胞源性泡沫细胞,分析干扰α-COP表达对泡沫细胞胆固醇流出率和细胞总胆固醇水平的影响,通过激光共聚焦显微镜观察细胞内脂肪分化相关蛋白(ADFP)和胆固醇探针FillipinⅢ表达的改变。查询GEO数据库,分析不同颈动脉粥样硬化斑块组织中α-COP的表达水平差异。结果与Ac-LDL组比较,apoA-1组泡沫细胞胆固醇流出率明显升高[(9.77±0.79)%vs.(2.74±0.37)%,P<0.001];油红O染色显示,apoA-1组泡沫细胞内脂质蓄积较Ac-LDL组减少。apoA-1组泡沫细胞内α-COP mRNA相对表达水平较Ac-LDL组升高(P<0.001),α-COP蛋白表达水平也升高(P<0.001)。荧光强度分析表明,apoA-1组α-COP的平均荧光强度较Ac-LDL组升高。shRNA敲低α-COP表达水平后,与Scr组比较,α-COP shRNA组泡沫细胞内apoA-1介导的胆固醇流出率降低(P<0.05),总胆固醇水平增加(P<0.001)。与Scr组比较,α-COP shRNA组泡沫细胞内ADFP的红色荧光强度增加(P<0.05),FilipinⅢ的蓝色荧光强度也增加(P<0.01)。GEO数据库结果表明,颈动脉粥样硬化斑块组织中α-COP mRNA表达水平低于正常动脉组织,且晚期颈动脉粥样硬化斑块组织α-COP mRNA表达水平低于早期颈动脉粥样硬化斑块组织(P<0.05)。结论αObjective To establish a human monocytic THP-1 macrophage-derived foam cell model and analyze the role of theαsubunit of coatomer proteinⅠ(α-COP)in apolipoprotein A-1(apoA-1)-mediated cholesterol efflux from foam cells.Methods THP-1 cells were induced to adhere using PMA(PMA group),followed by treatment with acetylated low-density lipoprotein(Ac-LDL)to generate macrophage-derived foam cells(Ac-LDL group).Subsequent incubation with apoA-1 formed the apoA-1 group.Cholesterol efflux rates mediated by apoA-1 and intracellular lipid accumulation were quantified through liquid scintillation counting and oil red O staining.The expression patterns ofα-COP were systematically analyzed using quantitative real-time PCR(qPCR),Western blotting,and laser scanning confocal microscopy.THP-1 macrophage-derived foam cells mediated by apoA-1 were transduced with scrambled shRNA(Scr group)orα-COP-specific shRNA lentivirus(α-COP shRNA group)to assess the effects ofα-COP knockdown on cholesterol efflux efficiency and total cellular cholesterol content.Subcellular localization of adipopoilin(ADFP)and cholesterol probe FilipinⅢstaining patterns were visualized via laser scanning confocal microscopy.Bioinformatics analysis ofα-COP expression profiles in carotid atherosclerotic plaques was performed using datasets from the GEO database.Results Compared with the Ac-LDL group,the cholesterol efflux rate in the apoA-1 group was significantly increased[(9.77±0.79)%vs.(2.74±0.37)%,P<0.001].Oil red O staining demonstrated reduced lipid accumulation in foam cells of the apoA-1 group compared with the Ac-LDL group.The relative mRNA expression level ofα-COP in the apoA-1 group was significantly higher than that in the Ac-LDL group(P<0.001),with corresponding elevation in protein expression(P<0.001).Fluorescence intensity analysis revealed increased mean fluorescence intensity ofα-COP in the apoA-1 group compared with the Ac-LDL group.Followingα-COP knockdown by shRNA,the apoA-1-mediated cholesterol efflux rate was significantly dec

关 键 词：外被体蛋白Ⅰ 载脂蛋白A-1 泡沫细胞 胆固醇流出 

分 类 号：R541.4[医药卫生—心血管疾病]