Effects of High-density Lipoprotein 1 on the Formation of Foam Cells from Human Monocytes-derived Macrophages
|School||Southern Medical University,|
|Course||Cardiovascular within science|
|Keywords||High-density lipoprotein Subclasses Macrophages Foam cells Cholesterol efflux|
Research background has been confirmed serum high-density lipoprotein (HDL) has anti-atherosclerosis (AS) and cardioprotective effects. HDL is composed by a variety of sub-categories with different particle properties and physiological function of different subclasses play a different role in the prevention of coronary heart disease (CHD) is important in the overall effect of HDL anti-AS. However, the composition of HDL subclasses and particle characteristics remain unclear, clinical HDL subclasses can not adjust the RCT and get significant treatment effect. HDL composition of the complex heterogeneity, due to the special structure of the particles, and thus how to detect HDL subclasses to analyze the state of the reverse cholesterol transport (RCT), and HDL subclasses in particular, is a new type of physical and chemical structure and physiological function of depth, is the focus of current research and difficult. In our previous study, by the segment concentration polyacrylamide gel electrophoresis (sd-PAGE) of the new method, the SBB prestained serum sync separation zone 11 to 13, respectively, are two kinds of free fatty acid (ALB- FFA 1-2 ), HDL subfractions (HDL 1-5 ) phenotype of low density lipoprotein (LDL), intermediate density lipoprotein (IDL), and very low density lipoprotein (VLDL), and chyle stranded at the origin of the fine particles (CM). Sd-PAGE new method not only serum CM, VLDL, IDL, LDL and HDL districts can separate and can be further sub-categories separation of each component, especially 5 subcategories new type of HDL (including HDL 5 ). In a previous study, we found that HDL 1 showed all the sd-PAGE experiments separating gel isolated sub-band, indicating that it is a relatively new independent component. This is a new phenomenon, and its conduct in-depth studies have important academic value and clinical significance. The aim of the present study was designed to use a new method of sd-PAGE separated from the serum with the preparation of HDL special subclasses of HDL 1 , from the observation of human peripheral blood monocyte-derived macrophage foam cell level the impact, to explore the role of HDL anti-atherosclerotic, and thus provide clues for its conduct in-depth research from the molecular level and the receptor level. Method 1 Preparation and oxidative modification of low density lipoprotein (LDL): disposable density gradient ultracentrifugation isolated from plasma LDL by agarose gel electrophoresis to identify the modified Lowry method for protein quantitation. Cu 2 sup> induced its oxidative modification, thiobarbituric acid value (TBARS) assay the degree of oxidation. 2, the separation and preparation of the serum HDL 1 : First with Sudan Black B (SBB) serum prestained Sectional concentration polyacrylamide gel electrophoresis (sd-PAGE) method sync separator various lipoprotein fractions of serum LDL, HDL and its subclasses using self elution, concentration device was prepared in quantitative serum HDL 1 . Using a modified method of Lowry its protein quantitation, biochemical enzymatic and turbidimetry detection HDL 1 solution content of apoA-I. 3, Preparation of human peripheral blood mononuclear cells: the density gradient centrifugation method and the plastic adsorption method to isolated mononuclear cells from the peripheral blood of a subject. Trypan blue exclusion staining and flow cytometry separation of cell survival and cell purity. 4, foam cell model 4.1 monocytes into macrophages: mononuclear cells to 50 nmol / L phorbol ester (PMA) induced 48 h, to make it into macrophages. Light microscope for morphological observation. 4.2 macrophages into foam cells: macrophages and 80 mg / L of oxidized low-density lipoprotein (ox-LDL) were incubated for 24 h into foam cells. Oil red O staining observed by light microscopy, transmission electron microscopy, and the determination of intracellular cholesterol content and protein content of the cell model of morphological and biological identification. 4.3 cell viability detection: using trypan blue exclusion staining cell activity and cell viability was measured in the experimental stage, observe different concentrations of HDL of 1 , as well as the same concentration of HDL 1 < / sub> adverse effects on cultured cells in the presence of toxic different time. 5, intracellular cholesterol and protein content determination: Determination of cholesteryl ester Enzymatic determination of intracellular cholesterol content, respectively, total cholesterol (TC) and free cholesterol (FC) content enzymatic assay kit to measure intracellular cholesterol content, TC subtracted FC represents the content of cholesterol ester (CE); using the modified Lowry method to the content of the determination of the intracellular protein (Pro), the final calculation of the cells CE / TC ratio and TC / Pro ratio. 6, HDL 1 of macrophage cholesterol and protein content: experimental cells divided into a control group and the experimental group, in which the control group, a blank control (RPMI-1640 group), positive control ( ox-LDL group) as well as standard control (of apoA-I of ox-LDL group) of the three groups, the experimental group cells first to 80 mg / L ox-LDL at different concentrations (0.1,1.0,10.0 mg / L) of HDL intervention 24 h, cholesteryl ester determination Enzymatic determination of intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) and other indicators to determine HDL 1 lower intracellular optimal concentration ratio of TC / Pro to make the relationship between the dose-response curve; macrophages to HDL 1 optimal concentration of pretreatment 1 h after adding 80 mg / L ox-LDL were incubated at different times 0,6,12,24 h. Detection indicators to determine HDL 1 to reduce intracellular ratio of TC / Pro best time to make aging curve. 7 Statistical analysis: The experimental results are used to the differences between the groups were analyzed by univariate analysis of variance (ONE WAY ANOVA), and then the LSD test Mean ± SD; unequal variances nonparametric rank sum test. Statistical analysis was performed using SPSS 10.0 statistical software, and α = 0.05 level of inspection. 1, the separation of serum lipoproteins: sd-PAGE SBB prestained serum synchronization separation zone 13, respectively for the two kinds of free fatty acids (AL-FFA 1-2 ) , five kinds of HDL subfractions (HDL 1-5 , where HDL 1 the HDL 5 its new type), three kinds phenotype low-density lipoprotein (LDL), intermediate density lipoprotein (IDL) and very low density lipoprotein (VLDL), and retention at the origin chylomicrons (CM). 2 cell model 2.1 monocytes into macrophages: induced by 50 nmol / L PMA for 48 h light microscope by the circular of the majority of monocytes into polygonal shape, and tend to clump adherent described mononuclear cells have been differentiated into the typical morphology of macrophages. 2.2 macrophages into foam cells: macrophages and 80 mg / L ox-LDL were incubated for 24 h after oil red O staining observed by light microscopy and transmission electron microscopy observation cell memory in a large number of red-stained lipid particles more lipid vacuoles, cholesterol Determination found that intracellular CE / TC> 60%, in line with the biological definition of foam cells. 3, HDL 1 on macrophage foam: HDL 1 role after treated with different concentrations, macrophages TC / Pro ratio with HDL 1 < / sub> of increasing concentrations of the dose dependent decrease (P <0.01), and macrophage foaming degree (CE / TC ratio) with reduced HDL 1 concentration increased, and in the case of a certain concentration, the ratio of intracellular TC / Pro with the HDL 1 treatment time was reduced time-dependent manner (P <0.01), compared with the blank, positive control group, were There are significant differences (P <0.05). Conclusion 1, the the Sd-PAGE electrophoresis new method not only can the CM, VLDL, IDL, LDL and HDL serum lipoprotein fractions separating gel clearly separate, and can be further sub-categories separation of each component, in particular, prominent is the the 5 subcategories HDL (HDL 1 , HDL , 5 and other for its new type). Separation of serum lipoprotein subfractions and past numerous methods, this method has a significant performance advantage. 2, in the process of macrophage foam, of HDL intervention intracellular TC / Pro ratio clear dose-response and time-effect relationship exists, that HDL 1 by lower levels of intracellular TC intracellular TC / Pro ratio and CE / TC ratio decreased to some extent reduce the degree of macrophage foam. The preliminary experiments confirmed HDL 1 inhibit macrophage foam cells function, and thus play a role in HDL anti-atherosclerotic process.