三磷腺苷合酶抑制因子1基因敲除对小鼠巨噬细胞线粒体功能和三磷腺苷水平的影响

Effects of adenosine triphosphate synthase inhibitor 1 gene knockout on the levels of adenosine triphosphate and mitochondrial function in mouse macrophages

目的探讨三磷腺苷合酶抑制因子1(ATPIF1)基因敲除对小鼠巨噬细胞内三磷腺苷(ATP)水平及线粒体功能的影响。方法选取5只野生型C57BL/6小鼠为WT组,5只ATPIF1基因敲除小鼠为KO组。处死小鼠后,提取小鼠骨髓细胞进行培养,并诱导其分化成熟为骨髓来源巨噬细胞(BMDM)。在未加脂多糖(LPS)的基础状态及LPS刺激2、4h后收集细胞,应用ATP检测试剂盒检测细胞内ATP水平,应用细胞能量代谢分析仪检测细胞氧消耗率,采用实时荧光定量聚合酶链反应检测细胞内三羧酸循环中关键酶丙酮酸激酶(PKm)、异柠檬酸脱氢酶(IDH)、柠檬酸合酶(CS)、α-酮戊二酸脱氢酶(OGDC)mRNA相对表达量。结果与WT组比较,在基础状态下KO组小鼠BMDM内ATP水平、基础氧耗量、用于ATP合成的氧耗量、最大氧耗量及PKm、IDH、CS、OGDCmRNA相对表达量显著增加(P<0.05,P<0.01);LPS刺激2、4h后,KO组小鼠BMDM内ATP水平、基础氧耗量、用于ATP合成的氧耗量和最大氧耗量及CS、OGDCmRNA相对表达量均显著增加(P<0.05)。结论ATPIF1基因敲除可增加小鼠BMDM内ATP水平,并提高三羧酸循环相关酶mRNA表达和线粒体氧化磷酸化水平。

Objective To investigate the effect of adenosine triphosphate synthase inhibitor 1 (ATPIF1) gene knockout on adenosine triphosphate (ATP) level and mitochondrial function of mouse macrophages. Methods Five wild type C57BL/6 mice were selected as WT group,and five ATPIF1 gene knockout mice were selected as KO group.After mice were sacrificed,bone marrow cells were cultured and induced to differentiate into bone marrow-derived macrophages (BMDM).Then cells were collected at the basic state without lipopolysaccharide (LPS) and at 2,4 hours after LPS stimulation,then ATP level in the BMDM was detected by ATP assay kit;cell oxygen consumption rate was measured by cell energy metabolism analyzer.The relative mRNA expression levels of key enzymes in the cell tricarboxylic acid cycle, including pyruvate kinase (PKm),isocitrate dehydrogenase (IDH),citrate synthase (CS) and α- ketoglutarate dehydrogenase (OGDC) was detected by real-time fluorescence quantitative polymerase chain reaction. Results Compared with the WT group,in the basic state,the ATP level,basic oxygen consumption ,oxygen consumption for ATP synthesis,maximum oxygen consumption,and the relative mRNA expressions of key enzymes PKm,IDH,CS and OGDC were significantly increased ( P <0.05, P <0.01).At 2,4 hours after LPS stimulation,the ATP level,the basic oxygen consumption,oxygen consumption for ATP synthesis, maximum oxygen consumption and the mRNA expression levels of CS and OGDC of tricarboxylic acid cycle genes in the KO group were significantly higher compared with those in the WT group( P <0.05). Conclusion ATPIF1 gene knockout increased the ATP level in the BMDM of mice,and increased the expression of mRNA of key enzymes of the tricarboxylic acid cycle and increased the level of mitochondrial oxidative phosphorylation.