巨噬细胞荷脂过程中脂肪分化相关蛋白与酰基辅酶A:胆固醇酰基转移酶1及中性胆固醇酯水解酶相互结合
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国家自然科学基金(30971268)


Interaction of the Adipophilin with Acyl-coenzyme A:Cholesterol Acyltransferse 1 and Neutral Cholesterol Ester Hydrolase in Lipid-loaded Macrophages
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    摘要:

    目的探讨脂肪分化相关蛋白是否与酰基辅酶A:胆固醇酰基转移酶1或中性胆固醇酯水解酶相互结合。方法50mg/L氧化型低密度脂蛋白孵育RAW264.7细胞0、0.5、1、3、6h,使用逆转录-聚合酶链反应及蛋白免疫印迹分析技术检测脂肪分化相关蛋白、酰基辅酶A:胆固醇酰基转移酶1和中性胆固醇酯水解酶的mRNA及蛋白的表达;应用免疫共沉淀技术检测脂肪分化相关蛋白与酰基辅酶A:胆固醇酰基转移酶1或中性胆固醇酯水解酶是否相互结合。结果随着氧化型低密度脂蛋白与RAW264.7细胞孵育时间的延长,逆转录-聚合酶链反应和蛋白免疫印迹分析显示,脂肪分化相关蛋白、酰基辅酶A:胆固醇酰基转移酶1和中性胆固醇酯水解酶的mRNA和蛋白质随着时间的延长而增多,与0h相比,差异有显著性(P<0.05,n=3)。免疫共沉淀实验显示,RAW264.7细胞与氧化型低密度脂蛋白孵育0、0.5、1、3h时脂肪分化相关蛋白与酰基辅酶A:胆固醇酰基转移酶1结合,6h时脂肪分化相关蛋白与酰基辅酶A:胆固醇酰基转移酶1不结合;孵育0、0.5、1h时脂肪分化相关蛋白与中性胆固醇酯水解酶不结合,3、6h时与中性胆固醇酯水解酶结合。结论在荷脂的RAW264.7细胞中脂肪分化相关蛋白与中性胆固醇酯水解酶及酰基辅酶A:胆固醇酰基转移酶1相互结合。脂肪分化相关蛋白与酰基辅酶A:胆固醇酰基转移酶1和中性胆固醇酯水解酶在细胞内脂质代谢中可能协同作用。

    Abstract:

    Aim To explore whether adipophilin interact with acyl-coenzyme A:cholesterol acyltransferse 1 (ACAT1) and neutral cholesterol ester hydrolase (NCEH) in lipid-loaded RAW264.7 cell induced by oxidized low density lipoprotein (ox-LDL).Methods RAW264.7 cells were incubated with 50 mg/L ox-LDL for different time.The expression of mRNA and proteins of adipophilin,ACAT1 and NCEH were detected by semi-quantitative reverse transcription-polymerase chain reaction and western blot respectively.Interactions between adipophilin and ACAT1 or NCEH were detected by co-immunoprecipitation.Results As the incubation time of ox-LDL was extended in RAW264.7 cells,the expression of mRNA and proteins of adipophilin,ACAT1 and NCEH were significantly increased compared with 0 h group (P<0.05,n=3).Co-immunoprecipitation showed that there were interactions between adipophilin and ACAT1 in RAW264.7 macrophages with ox-LDL treatment at 0,0.5,1 h and 3 h,and no interactions at 6 h.There were not interactions between adipophilin and NCEH in RAW264.7 macrophages with ox-LDL treatment at 0,0.5 h and 1 h,and interactions at 3 h and 6 h.Conclusions There were interactions between adipophilin and ACAT1 or NCEH in RAW264.7 macrophages with ox-LDL treatment.It suggests that adipophilin may have synergistic effects with ACAT1 and NCEH in lipid-loaded RAW264.7 cell.

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乔运成,彭娟,刘志强,郭东铭,袁中华.巨噬细胞荷脂过程中脂肪分化相关蛋白与酰基辅酶A:胆固醇酰基转移酶1及中性胆固醇酯水解酶相互结合[J].中国动脉硬化杂志,2014,22(12):1201~1206.

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  • 收稿日期:2013-12-02
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