缺氧条件下巨噬细胞移动抑制因子对肉鸡心肌脂肪酸代谢的影响

摘要

旨在探讨缺氧条件下巨噬细胞移动抑制因子(MIF)对肉鸡心肌脂肪酸代谢的影响.取9～12日龄鸡胚,采用胰蛋白酶和Ⅱ型胶原酶联合消化法分离培养心肌细胞,镜下观察细胞形态及活力,并对其进行免疫细胞化学鉴定.以低氧诱导因子1α(HIF-1α)为缺氧指标,化学缺氧模拟剂氯化钴(CoCl2)处理鸡胚心肌细胞,构建细胞缺氧模型.为了确认MIF与磷酸化腺苷酸活化蛋白激酶(p-AMPK)的关系,在缺氧模型上用MIF抑制剂(ISO-1)阻断MIF,观测p-AMPK是否随MIF阻断而变化.利用Western bolt检测脂肪酸代谢关键酶脂肪酸转运酶(FAT/CD36)、磷酸化乙酰辅酶A羧化酶(p-ACC)及肉毒碱棕榈酰转移酶(CPT-1 A)是否在缺氧状态下发生变化.结果:经差速培养的鸡心肌细胞纯度达90％以上;镜下观察,细胞呈梭形和不规则形,并可自发节律性搏动,频率80～130次·min-1.Western bolt检测显示,经600 μmol·L-1 CoCl2处理心肌细胞24 h,HIF-1α表达较正常对照组极显著升高(P＜0.01);100 μmol· L-1 ISO-1阻断MIF后,MIF及p AMPK表达量均明显降低(P＜0.01).与正常组相比,缺氧组心肌MIF、p-AMPK、FAT/CD36、p-ACC和CPT 1A表达均极显著升高(P＜0.01);ISO阻断组心肌各目的蛋白质的表达较缺氧组均极显著下降(P＜0.01).缺氧条件下,心肌自分泌MIF增多,通过激活AMPK促进脂肪酸相继进入细胞质和线粒体,加强氧化供能,为缺氧提供代偿性保护.%The aim of this study was to investigate the effects of macrophage migration inhibitory (MIF) on metabolism of Free-Fatty-Acid in broiler chicken cardiocytes in hypoxia.Firstly,chicken embryonic cardiomyocytes were isolated by trypsin and type Ⅱ collagenase,and cultured in DMEM,then identified by α-actinin immunocytochemistry.Secondly,hypoxia model of chicken cardiocyte induced by cobalt chloride was established,which was identified by the hypoxia sign protein,HIF-1α Thirdly,the MIF blocker ISO-1 was used to test the relationship between MIF and p-AMPK.The expression levels of FAT/CD36,p-ACC and CPT-1A in cardiomyocytes were determined by western blot during hypoxia and normoxia.Results were as follows:The chicken cardiocytes,above 90％ purity,showed spherical and polygon shape in vivo,and they beat spon taneously about 80-130 times per minute.HIF-1α expression reached the highest at 24 h with the 600 μmol · L-1 CoCl2.The p-AMPK levels decreased significantly (P＜0.01) along with the MIF which was blocked by 100 μmol · L-1 ISO-1.The levels of MIF,p-AMPK,FAT/CD36,pACC and CPT-1A were all increased significantly (P＜0.01) in the hypoxia group compared with the normal one,while the levels of the target proteins were all decreased significantly (P＜0.01) in the inhibitor group compared with the hypoxia one.MIF,a myocardial autocrine factor,plays an important protective role in the fatty acid adapted metabolism of chicken cardiomyocytes during hypoxia by activating the AMPK signal pathway.