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

Abstract

Abstract: 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.

Key words: Very low density lipoprotein, Twodimensional fluorescence difference gel electrophoresis, Foam cell, Proteomics

LU Yanjun;TIAN Jun;DI Yong;ZONG Yiqiang. Two-dimensional fluorescence difference gel electrophoresis analysis for VLDLinduced THP-1 foam cell formation[J]. , 2012, 27(1): 50-56.

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