Protemoic Analysis Of The Triglyceride-rich Very Low Density Lipoprotein-laden Foam Cell

It has been reported that hypertriglyceridemia is a risk factor for coronary heart disease. Atherosclerogenesis in hypertriglyceridaemic individuals is associated with the increased concentrations of VLDL and VLDL-associated remnant particles.In China, the people diet characterize low-fat and high glucose, the level and composition of lipids are different from the crowd with the West, the lipid disorders within the endogenous triglyceride (TG) increased is the most prevalent. TG is most in triglyceride-rich lipoprotein, while more than 90% the latter is VLDL, thus elevated TG in fact reflects the increase in VLDL. VLDL is a large lipoprotein particle that is composed of triglycerides, free and esterified cholesterol, phospholipids, and apolipoproteins, VLDL with the small diameter can enter vascular endothelium and cause lipid accumulation in macrophages.The macrophage-derived foam cells are the characteristics of pathological-inflammation cells occuring within the AS plaque. More traditional researchs focused on foam cell induced by cholesterol-rich lipoprotein oxidizd LDL. Because of most studies work on the proteins related to the formation of foam cells, which the function is regulating lipid metabolism, such as the receptor intaking lipid, LRP, LDLR, MSR, CD36 and so on, or related to the intracellular accumulation or efflux studies, and research studies are mostly point to point, with little regard whether the other pathophysiological changes have occured in macrophages after phagocytosis lipoprotein. It has been reported that the foam cells containing large amounts of lipids in the latter progress atherosclerotic prone to apoptosis.Thus, the more complex changes in the proteome must have occured in macrophages after the phagocytosis a large number of lipids.Proteomics point of view of the overall intracellular protein expression levels or modification of the state of change, to understand the interaction between proteins and the link,to explore the laws of life activities and its important pathophysiological nature at the protein level.At present, the proteomics research on foam cell has been undertaken, mainly concentrating in cholesterol-rich foam cell.However, the proteomics research on triglyceride-rich foam cells has not been reported.In this study, the cultured THP-1 cells were treated with phorbol ester (PMA) to induce for 48 hours, the cells were differentiated into macrophages.Extracting of LDL and VLDL by density gradient centrifugation, then 50μg/ml VLDL or oxidized-LDL after quantitation was used to incubate the differentiated macrophages for 48 hours.Oil red staining showed a large number of lipid accumulation in macrophages incubated with VLDL. Further determination of intracellular lipid components, the results suggest that the foam cells incubated with VLDL accumulate mainly in triglycerides, the triglyceride levels are 5.6-fold in contrast to the PBS-treated control macrophages.The experimental results showed that in vitro cultured macrophages can form foam cells after incubation with VLDL, and the lipid composition is major as triglycerides.Next, we established triglyceride-rich foam cells of two-dimensional map by two-dimensional electrophoresis. The results showed that over 1000 spots could be detected in each gel, the isoelectric point of most proteins was in the PH 5-7.114 protein spots were increased and 101 protein spots were down-regulated in VLDL-treated macrophage fom cells compared to the proteins in PBS-treated macrophage. Further, in order to identify the protein group, the protein spots were excised by hand, and then degraded by trypsin, the enzyme fragment were analysied by MALDI-TOF-MS to obtain protein peptide mass fingerprint (peptide mas fingerprint, PMF), then were searched in genomics and proteomics databases to achieve a qualitative identification of the proteins.Of which 14 different protein spots were identified by mass spectrometry, which includes the ADRP related to lipid droplets formation in foam cells, phosphoglycerate conjugation enzymes involved in glycolysis and the protein participating in oxidative stress.Meantime, the expression of some proteins was verified by Western Blot, and the results are consistent with the proteomic results, while their mRNA level was detection by RT-PCR, which also has changed. So in the process of formation foam cells, not only a large number of lipid accumulation in macrophage, but also accompanied other complex biological changes.Emerging fluorescence difference gel electrophoresis (difference in-gel electrophoresis, DIGE) technology can label the different specimens with different fluorescent dyes, the samples are mixed and seperated by isoelectric focusing and SDS-PAGE electrophoresis at the same time. Because of its good repeatability, high sensitivity, as it is now one of the tools of quantitative proteome research. However, the applications on their atherosclerosis have not been reported. On the basis of two-dimensional map of triglyceride-rich foam cells in previous study,50p,g of total cellular protein extracted in each group were used to be labled with Cy3 or Cy5,Cy2 as internal standard, captured the images with Typhoon 9410 scanner. Higher quality two-dimensional maps were eatablised, the matching rate of gel were 93%,94 protein spots were increased and 78 protein spots were down-regulated in VLDL-treated macrophage fom cells compared to the proteins in PBS-treated macrophage (1.5 fold).Fluorescence two-dimensional difference gel electrophoresis has a higher sensitivity, the analysis of differences in the protein are more reliable.In conclusion, the two-dimensional electrophoresis technology was used to carry out a proteome research for triglyceride-rich lipoprotein-induced foam cell in this study. A triglyceride-rich foam cells of the two-dimensional map was established by two-dimensional electrophoresis technology; 114 protein spots were increased and 101 protein spots were decreased in the foam cells incubated with VLDL with the analysis of Image master 6.0 software;14 proteins, including ADRP related to triglyceride accumulation in foam cells, Phosphoglycerate mutase, enolase related to glucose metabolism and so on, were identified with mass spectrometry. With the analysis of functions of these proteins, suggesting that VLDL not only affects the lipid accumulation in foam cells, may also affect other biological action (glucose metabolism, reactive oxygen species generation, etc.)in foam cells;Initially, the two-dimensional fluorescence difference gel electrophoresis technology were used for triglycerides-rich foam cells proteomic research, the map of two-dimensional fluorescence difference gel electrophoresis has been established.