| Atherosclerosis(AS) is a serious disease to human health. Coronary atherosclerotic heart disease is one of the biggest endanger. Especially with the aging of our society, our country at coronary heart disease (CHD) incidence and prevalence rates are increasing.Coronary heart disease is impacted by many factors. Variety of factors plsy different roles in coronary heart disease in different aspect. The main risk factors of coronary heart disease in general can be divided into immutable factors (such as gender, age, family history of coronary heart disease, etc.) and can change the factors or environmental factors (such as dyslipidemia, hypertension, smoking, diabetes and impaired glucose tolerance).Above all obesity, less physical activity, A-type personality, infection also important risk factor for CHD.Dyslipidemia, especially high cholesterol is a recognized risk factors.A large number of studies have shown that atherosclerosis happen have much relationship to low-density lipoprotein (LDL) and oxidative modification low-density lipoprotein. Natural LDL contains a large number of unsaturated fatty acids are prone to self-oxidation. In recent years, studies suggest that, LDL promote atherosclerosis development happen mainly through the role of ox-LDL to achieve. Another component of cholesterol, high density lipoprotein cholesterol also can be oxidized into oxidized high density lipoprotein (ox-HDL).When HDL became ox-HDL ,it also have a strong atherogenic effect, and lost its role of anti-AS.Cholesterol is an important lipid material in vivo biology. On the one hand, cholesterol is the basic component of biofilm;on the other hand, cholesterol is a bile acid, steroids and other physiologically active substances in raw materials. However, high concentrations of cholesterol can increase atherosclerosis and heart disease.So the concentration of cholesterol in the body is strictly regulated in order to ensure the normal functioning of biology.At the same time does not affect the body's health.The current study shows that high-density lipoprotein play a protective role on the cardiovascular system. This role is through the reverse cholesterol transport (RCT) mechanism to achieve. HDL plays the role of porters in this process.ATP binding cassette transporter A1 (ABCA1) can elevate HDL-C and increase RCT process. ABCA1 transport cholesterol components to HDL-C in RCT. HDL and ABCA1 plays a key role in cholesterol transportation. Research has shown that Ox-HDL can cause atherosclerosis. In this study, we try to intervent human macrophage with ox-HDL and ox-LDL,than detect the expression of ABCA1 mRNA. Explore the relationship between ox-HDL and ox-LDL with ABCA1.Sterol regulatory element binding proteins 2(SREBP-2) is one of important nuclear transcription factors, Sterol regulatory element bindingproteins SREBPs are a family of membrane bound transcription factors and play a fundamental role in both cholesterol metabolism SREBP-2 bind to sterol response elements in the enhancer/promoter region of target genes dedicated to the lipid synthesis, and activate their transcription1 So SREBP-2 are involved in the regulation of lipid homeostasis. In this study,we explore the relationship between oxidized LDL and oxidized HDL with SREBP-2, than discuss the role of ox-LDL ox-HDL and SREBP-2 in atherosclerosis.ObjectiveTo establish the monocyte-derived macrophage model derived from peripheral blood of coronary heart disease patients and normal control people, and then used different concentrations of ox-LDL and ox-HDL to interfere these monocyte-derived macrophage. To observe the expression of SREBP-2 and ABCA1 gene of these cells explored to different concentrations of ox-LDL and ox-HDL. And try to implicate the potent postulation underlining the development of coronary heart disease and SREBP-2 and ABCA1 expression of macrophage by ox-LDL and ox-HDL administrated.Methods1,Objectives: According to the diagnosis standards of "unstable angina and non-ST-segment elevation myocardial infarction diagnosis and treatment guidelines" published in 2007 by Chinese Medical Association and "ST-segment elevation myocardial infarction treatment guidelines -2004" published ACC / AHA in 2004,we selected studied objects.Patients of coronary artery disease were diagnosised by the medical history, electrocardiogram, myocardial enzymes, cardiac troponin I and coronary angiography. Total patients are 50 cases, 26 cases are male, 24 cases are female.Average age (60.12±8.36) years old. Upon inquiry, the select history, physical examination, laboratory tests (blood, urine, then conventional, blood lipids, blood glucose, liver and kidney function), electrocardiogram, echocardiography, X-ray examination and coronary angiography to exclude coronary artery disease,and normal 12 cases go to the control group. Six cases are male, 6 cases female, age 59.83±7.20 years old. In each group, sex and age have no significant difference (P> 0.05). Ethics Committee agreed this study.2,Experimental steps: 2.1 All selected patients underwent coronary angiography, according to the results of coronary angiography and clinical symptoms, electrocardiogram and myocardial enzyme changes,all patients are divided into 6 group: (1), 12 cases of normal control group; (2), non-intervention group of 10 coronary heart disease cases; (3) , 40m/L ox-LDL and 10 cases of coronary heart disease group; (4), 120mg/L ox-LDL group and 10 cases of coronary heart disease. (5), 40mg / L ox-HDL group and 10 cases of coronary heart disease; (6), 120mg / L ox-HDL group and 10 cases of coronary heart disease. Strict selection of cases registered general information,such as age, sex, blood lipids.2.2 Use density ultracentrifugation separate of LDL and HDL. Containing 10μmol/L CuSO4 in PBS solution to 37℃oxidation color from orange to white (6 hours), then PBS dialysis solution purification, filtration sterilization, BCA quantitaty lipoprotein, calmodulin concentration to 1g/L. Polyacrylamide gel electrophoresis identify ox-LDL and ox-HDL.2.3 Before coronary angiography, perform sterile operation, collect 20ml blood samples from the radial artery or femoral artery,then using Ficoll density gradient centrifugation separate peripheral blood mononuclear cells (PBMC).2.4 At 37℃, 5% CO2 incubator incubated mononuclear cells for 4 hours. Removed non-adherent lymphocytes, the remained were mononuclear cells. Purity of peripheral blood monouclear cells were detected by flow cytometry of CD14. Each group was stimulated by culture medium contained PMA of final concentration of 50 ng/ml for 48 hours. Cultivate groups with ox-LDL and ox-HDL for 48 h.2.5 After 48 h culture, collected macrophages, Trizol extraction of total RNA, UV spectrophotometer and agarose gel electrophoretic analysis purity.2.6 ABCA1 and SREBP-2 gene sequences were from PubMed. Using Primer Premier 5. 0 to design primer for ABCA1 and SREBP-2 cDNA amplification. The expression of ABCA1 and SREBP-2 in different groups detected by relative fluorescence quantitative RT-QPCR.3,Statistical analysis: Using SPSS 13.0 statistical software for statistical analysis.Measurement data use Mean±Standard Deviation. Normal distribution of data using one-way ANOVA statistics.Equal variances assumed multiple comparisons between groups using LSD method. Equal variances not assumed measurement data using Dunnett T3 test. P<0.05 had significant difference.Results1. Different groups of age, sex, had no statistically significant difference. Total Cholesterol (TC),Triglyceride (TG),LDL-C,HDL-C have statistical difference between Coronary heart disease group and normal control group (P<0.05). TC,TG and LDL-C of coronary heart disease group were higher than the normal control group. HDL-C of coronary heart disease group were lower than the normal control group. BMI,TC,TG,HDL-C and LDL-C between coronary heart disease group had no statistical difference.2. Isolated LDL and HDL. To produce oxidated lipoprotein.3.We have established a source of peripheral blood mononuclear cells macrophage model.4.The SREBP-2 and ABCA1 mRNA expression have no significant difference between the CHD group of non-intervention and the normal control group.5. Compared to the control group,the SREBP-2 expression between 40 mg/L ox-LDL and ox-HDL group increased significantly,and there is statistically significant difference (P<0.05). Compared to the control group,the SREBP-2 expression between 120mg/L ox-LDL and ox-HDL group had no significant difference,P=0.96. SREBP-2 expression between 40mg/L and 120mg/L ox-LDL intervention group has significant difference (P<0.05) , the same for 40mg/L and 120mg/L ox-HDL intervention group.6. ABCA1 expression between 40 mg/L ox-LDL group and the control group had significantly increase, the difference had statistical significance (P<0.05). ABCA1 expression between 120 mg/L ox-LDL group and the control group had significantly decrease, the difference had statistical significance (P<0.05).ABCA1 expression between 40 mg/L ox-LDL group and 120 mg/L ox-LDL group had significantly increase, and the difference had statistical significance (P<0.05). Compared to the control group , ABCA1 expression between 40 mg/L ox-HDL group and 120 mg/L ox-HDL group had significantly decrease, and the difference had statistical significance (T<0.05). The two intervention groups had significant difference (P<0.05).Conclusion1. High TC, high TG, high LDL-C, low HDL-C are important risk factors of coronary atherosclerosis.2. The expression of SREBP-2 and ABCA1 mRNA in macrophages derived from peripheral blood mononuclear cells between normal groups and patients with coronary artery disease had no significant difference. SREBP-2 and ABCA1 may not be an independent risk genes for coronary heart disease.3. Low concentrations of ox-LDL and ox-HDL can increase the expression of SREBP-2, high concentrations of ox-LDL and ox-HDL was neutral for the SREBP-2 expression, and had decrease tendency. Probably in atherosclerotic process, ox-LDL and ox-HDL may be the promoter of cholesterol synthesis mediated by SREBP-2.Sustained high concentrations of ox-LDL and ox-HDL may be reduced cholesterol synthesis mediated by SREBP-2.4. Low concentration of ox-LDL can promote the expression of ABCA1.That is mean in the early stage of AS low concentration of ox-LDL may stimulate the compensatory mechanism of reverse cholesterol transport mediated by ABCA1. In the decompensated stage of AS, high concentrations of ox-LDL can inhibit ABCA1-mediated reverse cholesterol transport. ox-HDL inhibited ABCA1 expression sustainably, and eventually leading to decrease reverse transport of cholesterol reduction mediated by ABCA1. However, this mechanism is unclear.
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