| BackgroundWith the development of society and the improvement of people’s living standard, the incidence of coronary heart disease is increasing year by year, at present coronary heart disease is one of the high incidence and risk disease in our country and around the world. As everyone knows, atherosclerosis lesions is the main basis of the coronary heart disease, however macrophage formating into foam cells, and too much cholesterol accumulated in the foam cells, plays a crucial role in the development of atherosclerosis. During the formation of atherosclerosis, the mononuclear cells adhered the damaging endothelial cell in the blood, the latter in monocyte chemotactic factor stimulation and various chemokines, migratiing to the vascular endothelium, further transformed into macrophages, the macrophage swallowed large amounts of oxidized low density lipoprotein (ox-LDL), caused imbalance in the cell, so that a large number of cholesterol accumulated in the cell, eventually the bubble formation of macrophage derived foam cells.Therefore, foam cell formation is the key link in the process of atherosclerosisCholesterol efflux is reverse cholesterol transport,which have capacity to discharge too much cholesterol in foam cells, to avoid the accumulation of excessive cholesterol in cells, prevent the formation of foam cells and the occurring of atherosclerosis. There are three transport pathway for cholesterol efflux to achieving, the ATP binding cassette transporter A1 (ABCA1) pathway plays a key role in the cholesterol transport process. Its main function is to combine with apolipoprotein Al, to formation of high density lipoprotein, promote cellular cholesterol efflux, binding the apolipoprotein Al form HDL, to avoid the accumulation of cholesterol in cells. In 1999, the first ABCA1 gene in a familia1-Tangier disease (high density lipoprotein deficiency) patients were found, officially due to ABCA1 gene mutation, loss of function, make the cholesterol efflux disorder, resulting in a large number of cholesterol intracellular accumulation, causing atherosclerosis occurrence. Therefore, ABCA1 has anti atherosclerosis function, has a protective effect on cardiovascular system.The study found that, various risk factors of many inflammatory factors, free radicals cause endothelial injury, not only make the monocytes to macrophages change this process accelerated, increase the phagocytic function of macrophage, and prompted the low density lipoprotein (LDL) into ox-LDL, Increased the formation of foam cells, lead to atherosclerosis,caused vascular intimal thickening, vascular stenosis formatting finally. But whether through the effection of reverse cholesterol transport pathways to the intervent the cholesterol metabolism in cells is not clear. Therefore, Study on the influence factors of these damage cholesterol transport and transport in foam cells pathway, contribute to insight into pathogenesis of atherosclerosis, which has the vital significance to the prevention and development of atherosclerosis in the future. Cystatin C (CysC) is one of the independent risk factor of atherosclerosis, which is currently recognized, it is a member of the cysteine protease inhibitor family, which was isolated and purified from the egg first USA scientists Anastasi who using chromatography in 1983, its molecular weight is only 13.3KD, which belongs to the small molecular weight and low molecular protein. The latest domestic and foreign research data show that, CysC is involved in the occurrence and development of cardiovascular diseases, which is one of the more related risk factors of atherosclerosis induced by now. Kestenbaum found that, the carotid artery intima media thickness in High CysC hyperlipidemia patients was significantly higher than that of normal serum CysC levels, and decrease the CysC level in serum, carotid artery intimal thickening degree was improved, therefore, CysC is involved in the pathogenesis of atherosclerosis, Sustained high level of serum CysC can continuous making the intimal thickening. Fraley found that, in hyperlipidemia, metabolic disorder of LDL patients, the serum CysC level increased significantly, think that serum CysC leve 1 has the synergistic effect on LDL concentration, The two factors not only participate in the occurrence and development of atherosclerosis, but also increased the extent of atherosclerosis. We have studied the role of CysC in rabbit after angioplasty restenosis formation and explore the mechanism. In the course of the experiment,48 New Zealand white rabbits were randomly divided into injury group (femoral artery puncture retention sheath tube parallel balloon injury), the treatment group (puncture after balloon injury in the abdominal aorta and local injection of CysC monoclonal antibody intervention) and control group (femoral artery puncture, retention sheath), each group have 16 rabbit, respectively in the preoperative, postoperative serum CysC level in different period of time.6 weeks after the operation, abdominal aorta smooth muscle actin staining, parallel HE and PCNA and other immunohistochemical staining, measuring neointimal area, luminal area, the residual stenosis rate, and analyze the correlation with serum CysC level. The results found that rabbit vascular atherosclerotic abdominal aorta after balloon injury, intimal hyperplasia occurs, vascular stenosis, and elevated serum CysC level, are in positive correlation (P< 0.01), the monoclonal antibody CysC intervention, serum CysC level decreased, atherosclerosis and intimal hyperplasia by inhibiting atherosclerosis, reduce vascular stenosis the degree of stenosis. Therefore, we believe that the high level of CysC, accelerate the progression of atherosclerosis, which aggravate the vascular stenosis, and CysC monoclonal antibody can antagonize the effect of CysC on arterial injury, delayed arterial stenosis, to the protective effect on blood vessels. CysC induced atherosclerosis possible mechanism is:during the oxidation process, CysC thiol to produce a variety of oxygen free radicals, such as hydrogen peroxide, superoxide anion, can accelerate the oxidation of LDL, oxidized LDL becomes ox-LDL, prompting the macrophage phagocytosis of large ox-LDL and foam into foam cells, occurrence, development process involved in atherosclerosis. The pathogenic mechanism, we are just inferences, not in-depth study.If CysC through the formation of foam cells. Effects the expression of intracellular cholesterol metabolism and transport protein ABCA1, caused the disturbance of cholesterol metabolism, eventually leading to the occurrence of atherosclerosis,the research in this area are rarely reported at home and abroad. This study aims to explore the metabolism of CysC of THP-1 (human acute monocytic leukemia cell line) affect the expression of cholesterol and transport protein ABCA1 derived foam cells, to study its pathogenic mechanism, providing the theoretical basis for the prevention and treatment of atherosclerosis.The experiment was divided into two parts, the first part is the preparation and identification of THP-1 derived foam cell, the second part is the effect of CysC on the expression of cholesterol metabolism and transport protein ABCA1 THP-1 derived foam cells. Only get THP-1 derived foam cells with stable property, to lay a solid foundation for the subsequent intervention experiment on expression of reverse cholesterol transport and transporter ABCA1, The first part focuses on the preparation process of THP-1 derived foam cells, method for identification of foam cells, including the phorbol 12-myristate 13-acetate (PMA) to induce the differentiation of THP-1 into the optimal conditions of macrophages and ox-LDL induced macrophage derived foam cells induced by the optimal concentration and induction time. The second part will be different co culture CysC concentration and foam cells, while adding CysC monoclonal antibody intervention, using ox-LDL, the content of cholesterol concentration detection in foam cells, determination of foam cell membrane and immune fluorescence detection method on transporter ABCA1 expression, and To explore the mechanism of CysC on the expression of cholesterol metabolism and transport protein ABCA1 THP-1 derived foam cells.Part â… Preparation and identification of the THP-1 derived foam cellsObjectiveMethods for the identification of the optimal preparation conditions of THP-1 derived foam cells and foam cells.Methods1. The preparation method of the main solution, including the RPMI-1640 culture medium, preparation of Coomassie brilliant blue staining solution, buffer PBS, oil red O solution and hematoxylin solution.2. Preparation method of ox-LDL.3. THP-1 cells culture method.4. Macrophage foaming.5. The process and identification method of THP-1 derived foam cell preparation.Results1.50ng/ml induced by PMA in THP-1 cells after 48h source formed typical macrophages (cells into irregular state, under the microscope into polygonal, prism and so on, by the suspended state to the adherent state).2.50ug/ml ox-LDL induced macrophage 48h foam into typical foam cells (cytoplasm into a bubble like change, increase total cholesterol concentration, intracellular cholesterol ester to total cholesterol content of more than 60%).ConclusionsThe preparation and identification process of THP-1 derived foam cell is a simple operation, foam cell model is stable, experimental method is scientific, reliable, provides a good experimental platform for the subsequent atherosclerosis related research, has laid a solid experimental basis.Part â…¡ Effects of CysC on the expression of cholesterol metabolism and transport protein ABCA1 THP-1 derived foam cellsObjective To observe the effects of different concentrations of CysC on the expression of cholesterol metabolism and transport protein ABC A1 THP-1 derived foam cells.MethodsThe group was divided into the control group and the experimental group, the control group is foam cells group, CysC was not join the control group to intervente. The experimental group were divided into 10ug/ml group,20ug/ml group,30ug/ml group,40ug/ml group,50ug/ml group and 30ug/ml +CysC monoclonal antibody group according to the CysC concentration, respectively 24h co cultured with different concentration of CysC and foam cells, oil red O staining, foam cell count, ox-LDL ELISA reaction determination of ox-LDL foam intracellular THP-1 source kit, determination of content of total cholesterol, THP-1 derived foam cells free cholesterol and cholesterol ester cholesterol oxidase endpoint detection method, and the foam cell membrane transport protein ABCA1 expression by immunofluorescence assay for determination of 30ug/ml group and 30ug/ml+CysC monoclonal antibody group, at the same time, compared with the control group.Results1. Different concentrations of CysC were incubated with THP-1 derived foam cells after 24h, oil red O staining, increase the formation of foam cells in the experimental group, were higher than the control group (P< 0.05), but the number of foam cell formation not with the concentration of CysC dose dependently, the number of 30ug/mlCysC group has the most number of foam cells, group 40ug/mlCysC and group 50ug/mlCysC group of foam cells was lower than that of 30ug/mlCysC group decreased, but the difference was not statistically significant (P> 0.05), the number of 30ug/ml+CysC monoclonal antibody group foam cells decreased obviously than the other concentration group (P< 0.05) compared with the control group, but no significant difference (P> 0.05).2. The ox-LDL content in the experimental group in foam cells was increased, there was statistically significant difference compared with the control group (P< 0.05), but its content is not with the dose dependent relationship of CysC concentration, group 30ug/mlCysC in foam cells of ox-LDL was the highest, 40ug/mlCysC group and 50ug/mlCysC group in foam cells decreased than in the 30ug/mlCysC group, but no statistical significance the difference (P> 0.05), the content of ox-LDL 30ug/ml +CysC monoclonal antibody group in foam cells decreased obviously than the other concentration group (P< 0.05) compared with the control group, but no significant difference (P> 0.05).3. The total cholesterol, free cholesterol and cholesterol ester content was increase in the experimental group in foam cells, there is a statistically significant difference compared with the control group (P< 0.05), but its content is not with the dose dependent relationship of CysC concentration, The total cholesterol, free cholesterol and cholesterol ester in 30ug/mlCysC content group was the highest, and 40ug/mlCysC group 50ug/mlCysC foam intracellular free cholesterol and cholesterol ester content decreased,than the 30ug/mlCysC group,but the difference was not statistically significant (P> 0.05),30ug/ml +CysC monoclonal antibody group in foam cells of total cholesterol, free cholesterol and cholesterol ester content decreased obviously than the other concentration group (P< 0.05), but the difference compared with the control group no statistical significance (P> 0.05).4. Immunofluorescence showed the expression of 30ug/mlCysC protein decreased obviously in ABCA1 foam cells group than in the control group (P< 0.05), the expression of 30ug/ml +CysC ABCA1 protein monoclonal antibody group foam cells was significantly stronger than that of 30ug/mlCysC group (P< 0.05), Compared with the control group have no significant difference (P> 0.05).Conclusions1. CysC hindered outflow THP-1 derived foam cells cholesterol, increased the intracellular accumulation of cholesterol, promote the formation of foam cells.2. CysC inhibited the expression of ABCA1 protein in THP-1 derived foam cell.3. CysC block cholesterol outflow by inhibited the expression of ABCA1 protein in THP-1 derived foam cell.4. CysC monoclonal antibody can promote the expression of ABCA1 protein in THP-1 derived foam cell, increased cellular cholesterol efflux, reduce the formation of foam cells, atherosclerosis, for the future development, the research and development of the related drugs intervention levels of CysC to the treatment of cardiovascular disease which provides a theoretical basis. |
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