运用双向荧光差异凝胶电泳分析VLDL致THP-1来源泡沫细胞的形成

Two-dimensional fluorescence difference gel electrophoresis analysis for VLDL-induced THP-1 foam cell formation

目的运用双向荧光差异凝胶电泳结合质谱技术寻找极低密度脂蛋白(VLDL)致诱导分化的THP-1细胞形成泡沫细胞中的关键蛋白质,以期揭示其致泡沫细胞的机制。方法 THP-1细胞经佛波醇脂(PMA)诱导分化为巨噬细胞,以磷酸盐缓冲液(PBS)孵育为空白对照组、VLDL孵育为实验组。提取细胞总蛋白后分别用荧光染料CY3和CY5标记,以所有样本等量混合作为"内标",用CY2标记,然后将实验组、空白对照组及内标混合后进行二维电泳,扫描图像后经分析的差异蛋白做基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)。采用逆转录-聚合酶链反应(RT-CPR)分析3种蛋白质(脂肪分化相关蛋白、烯醇化酶1、磷酸甘油酸变位酶1)mRNA的变化。结果实验组与空白对照组比较,表达量上调蛋白点在1.2倍以上有34个,表达量下调1.2倍以上有48个。通过软件筛选其中14个有差异的蛋白点做MALDI-TOF-MS,其中脂肪分化相关蛋白、热休克蛋白27、人电子转移味蛋白三维结构-链A、s100钙结合蛋白、人过氧化物酶-3-异构体c、辅酶-细胞色素C还原酶核心蛋白I、过氧化物酶烯酰辅酶A水合酶样蛋白、富马酸合酶-异构体d表达增高,烯醇化酶1、磷酸甘油酸变位酶1、环孢素A结合双肽甘氨酸-脯氨酸复合物、DMGDH蛋白、核内不均一核糖核蛋白L-异构体a、果糖胺3激酶-异构体b表达降低。脂肪分化相关蛋白mRNA表达明显上调,烯醇化酶1、磷酸甘油酸变位酶1 mRNA表达明显下调,与蛋白水平变化一致。结论荧光差异凝胶电泳能在整体上揭示VLDL在致泡沫细胞形成过程中蛋白质水平的变化,经鉴定的蛋白质可能在VLDL致泡沫细胞的形成中发挥重要的作用,为阐明VLDL作用巨噬细胞形成泡沫细胞的机制奠定了基础。

Objective To search for the key protein in the process of THP-1 foam cell formation induced by very low density lipoprotein（VLDL） through using two-dimensional fluorescence difference gel electrophoresis（DIGE） analysis and analyze the mechanism of foam cell.Methods The macrophage differentiation from THP-1 cells induced by phorbol ester（PMA）,incubated with phosphate buffer（PBS）as control group and incubated with VLDL as experimental group,respectively.After the extraction of total cellular protein labeled with fluorescent dye CY3 and CY5,the mixture of all samples were equalled as an ＂internal standard＂,labeled with CY2.The mixture of experimental group,control group and the internal standard was analyzed by DIGE and matrix-assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF-MS）.The 3 protein（adipose differentiation-related protein,enolase 1 and phosphoglycerate mutase 1） mRNA changes were analyzed with reverse transcription-polymerase chain reaction（RT-PCR）.Results Compared the experimental group with control group,there were 34 protein spots up regulated 1.2 times,and 48 protein spots down regulated 1.2 times.A total of 14 protein spots defined as ＂significant spots＂ were identified by MALDI-TOF-MS.The expressions of adipose differentiation-related protein,heat shock 27kDa protein 1,three-dimensional structure of human electron transfer flavoprotein to 2.1 A resolution-Chain A,S100 calcium binding protein A11,peroxiredoxin 3-isoform CRA_c,ubiquinol-cytochrome c reductase core protein I,peroxisomal enoyl-coenzyme A hydratase-like protein and fumarate hydratase-isoform CRA_d were increased.The expressions of enolase 1,phosphoglycerate mutase 1,cyclophilin A complexed with dipeptide Gly-Pro,DMGDH protein,heterogeneous nuclear ribonucleoprotein L-isoform CRA_a and fructosamine 3 kinase-isoform CRA_b were decreased.The mRNA expression of adipose differentiation-related protein was increased,and the mRNA expressions of enolase 1 and phosphoglycerate mutase 1 were decreased obviously with the levels of proteins.Conclusions DIGE has revealed a whole change in the process of VLDL-induced foam cell formation,and the protein identified may play an important role in the process,which as the ground work for clarifying the mechanism of VLDL-induced foam cell formation.