D4-F对THP-1巨噬细胞源性泡沫细胞ABCA1表达及胆固醇流出的影响
发布时间：2018-11-15 07:50
【摘要】： 目的:三磷酸腺苷结合盒转运体A(ATP-binding cassette transporter A1, ABCA1)在细胞内胆固醇流出中具有重要的作用。载脂蛋白A-1模拟肽D4-F是含有18氨基酸残基的小分子肽,其-两性螺旋基序是与脂质结合重要的结构基础。目前,动物实验证明,D4-F具有抗炎抗动脉粥样硬化的作用,在D4-F喂养的小鼠血液中,其三酰甘油和低密度脂蛋白胆固醇水平较正常组降低。在细胞试验中发现D4-F能够上调ABCA1蛋白质水平,但其具体的机制不清楚。 Cdc42是RhoGTP酶超家族成员,分子量为25KD。有研究表明Cdc42参与细胞内胆固醇的流出。丹吉尔病患者的成纤维细胞和巨噬细胞中Cdc42表达下调。apoA-1与ABCA1结合后,能够活化Cdc42。而本实验组研究已经证实apoA-1通过cAMP/PKA信号途径影响ABCA1蛋白质的表达及其介导的胆固醇的流出。基于这些研究,我们以THP-1巨噬细胞源性泡沫细胞为模型,探讨D4-F对ABCA1表达及其介导的胆固醇的影响,并进一步观察Cdc42/cAMP/PKA是否在这个过程中起作用。 第一部分D4-F对ABCA1表达及其介导的胆固醇流出的影响 目的: D4-F作为apoA-1的模拟肽,它是否影响THP-1巨噬细胞源性泡沫细胞中ABCA1表达及其介导的胆固醇流出?故本实验的目的是观察D4-F对THP-1巨噬细胞源性泡沫细胞中ABCA1表达及其介导的胆固醇流出的影响。 方法: THP-1单核细胞经PMA诱导贴壁后,加入oxLDL诱导其转化为泡沫细胞后,1.不同浓度(0μg/ml2μg/ml4μg/ml6μg/ml)D4-F处理细胞24小时; 2. 6μg/ml D4-F处理细胞不同时间(061224小时)和5μg/ml BSA处理细胞24小时;3. 6μg/ml D4-F与20μg/ml D4-F放线菌酮共处理细胞0,1,2小时。实时定量PCR和Western印迹分析法分别检测ABCA1 mRNA与蛋白质的表达;液体闪烁计数器检测细胞内胆固醇流出,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量。 结果: D4-F不影响ABCA1m RNA水平; 6μg/ml D4-F和D4-F处理细胞24小时, ABCA1蛋白质的表达至高峰;加入放线菌酮抑制蛋白质合成之后,D4-F能够明显减缓ABCA1蛋白质的降解;高效液相色谱法显示D4-F降低了细胞内总胆固醇,游离胆固醇和胆固醇酯的含量。液体闪烁法检测D4-F能够浓度依赖性和时间依赖性提高细胞内胆固醇及其磷脂的流出。 第二部分D4-F增加ABCA1表达及其介导胆固醇流出的机制探讨 目的:前期实验结果显示D4-F增加ABCA1蛋白质表达及其介导的胆固醇流出,故本实验主要探讨D4-F影响ABCA1蛋白质表达及其介导胆固醇流出的可能机制。 方法: THP-1单核细胞经PMA诱导贴壁后,加入oxLDL诱导其转化为泡沫细胞,1. 6μg/ml D4-F处理细胞0,15,20,25分钟后,观察PKA活性;2.转染PKA-siRNA后,6μg/ml D4-F孵育24小时;3. 1.0mM PKA特异性激动剂dBcAMP与6μg/ml D4-F孵育24小时;4. 20μg/ml D4-F放线菌酮与PKA-siRNA干扰或dBcAMP和6μg/ml D4-F处理细胞0,1,2小时;5. 6μg/ml和100μg/l腺苷酸环化酶抑制剂SQ22536或25mM Cdc42抑制剂Scramine B在37℃孵育30分钟;6. 6μg/ml D4-F处理细胞0,10,15,20分钟后,观察Cdc42活性。吸光度值检测PKA活性和Cdc42活性检测试剂盒检测Cdc42活性;液体闪烁计数器检测细胞内胆固醇流出;Western印迹分析法检测ABCA1蛋白质的表达和ABCA1磷酸化;酶联免疫试剂盒检测cAMP水平。 结果: D4-F激活PKA;PKA-siRNA能下调PKA蛋白质的表达,与对照组比较表达下调达85%;PKA-siRNA和dBcAMP均不影响D4-F增加ABCA1蛋白质的作用;PKA-siRNA,dBcAMP,SQ22536均能逆转D4-F引起的ABCA1介导的胆固醇流出增加;同时,SQ22536能逆转D4-F引起的cAMP水平,PKA活性及ABCA1磷酸化的增加;D4-F能影响Cdc42活性;Scramine B影响了D4-F引起的cAMP水平和PKA活性增加。 结论: 1 D4-F呈时间和浓度依赖上调ABCA1蛋白质表达; 2 D4-F可以通过Cdc42/cAMP/PKA途径增加ABCA1丝氨酸磷酸化和ABCA1介导的胆固醇流出。
[Abstract]:Objective: The expression of ATP-binding cassette transporter A (ABCA1) in the 3-phosphate-binding cassette transporter A (ATP-binding cassette transporter A1, ABCA1) plays an important role in the efflux of cholesterol in the cells. Apolipoprotein A-1 mimetic peptide D4-F is a small molecular peptide containing 18 amino acid residues, and the-amphoteric helical motif is a structural basis that is important for lipid binding. At present, animal experiments show that D4-F has the effect of anti-inflammatory and anti-atherosclerosis, and in the blood of the mice fed with D4-F, the level of triglyceride and low-density lipoprotein cholesterol is lower than that of normal group. It was found that D4-F can increase the level of ABCA1 protein in cell test, but its specific mechanism is not clear. Cdc42 is a member of the RhoGTP enzyme superfamily with a molecular weight of 25KD. It was found that Cdc42 was involved in the cell liner. The outflow of alcohol. Cdc42 in fibroblasts and macrophages of patients with Tangier disease After the binding of apoA-1 and ABCA1, C can be activated dc42. The study of the experimental group has demonstrated that apoA-1 has the effect of the cAMP/ PKA signaling pathway on the expression of ABCA1 protein and its mediated cholineralization. Based on these studies, we studied the effect of D4-F on the expression of ABCA1 and its mediated cholesterol, and further observed whether Cdc42/ cAMP/ PKA was in this process. The effect of the first part D4-F on the expression of ABCA1 and its medium Effect of guided cholesterol efflux: D4-F as an analog peptide of apoA-1, and whether it affects the ABC of the THP-1 macrophage-derived foam cells The purpose of this experiment was to observe the ABCA1 table in the source foam cells of the THP-1 macrophage by D4-F. Dup and its effect on cholesterol efflux. Methods: THP-1 monocytes were induced by PMA, and then added. After the induction of oxLDL to the foam cells, 1. The different concentration (0. mu.g/ ml? 2. mu.g/ ml? 4. mu.g/ ml? 6. m g/ ml) D4-F treated cells for 24 h; 2.6. mu.g/ ml D4-F for different time (0? 6? 12? 24 h); and 5. mu.g/ ml BSA treated cells for 24 hours; 3.6. mu.g/ ml D4-F and 20.mu. g/ ml D The expression of ABCA1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. The contents of total cholesterol, free cholesterol and cholesteryl ester in the cell were determined. The results showed that D4-F did not affect the level of ABC1m RNA, and the expression of ABCA1 protein reached the peak at 6. m u.g/ ml of D4-F and D4-F. After the inhibition of protein synthesis, the D4-F could significantly slow the degradation of the ABCA1 protein.; High performance liquid chromatography to show D4-F The content of total cholesterol, free cholesterol and cholesteryl ester in cells is reduced. The concentration of D4-F can be detected by liquid scintillation. The dependence and time-dependence increase the efflux of cholesterol and its phospholipids in cells. Two-part D4-F increased the expression of ABCA1 and its mechanism to mediate the outflow of cholesterol. The effect of D4-F on the expression of ABCA1 protein and its possible mechanism to mediate the outflow of cholesterol were mainly discussed. after the THP-1 monocytes were induced to adhere by PMA, oxLDL was added to induce it to be converted into foam cells, 1. 6. mu. g/ ml of D4-F treated cells for 0, 15, 20, 25 minutes, observed PKA activity; 2. After the PKA-siRNA was transfected, 6. mu.g/ ml of D4-F was incubated for 24 hours; 3. 1. 0mM PKA specific agonist dBcAMP incubated with 6. mu.g/ ml D4-F for 24 hours; 4.20. mu.g/ ml of D4-F, and PKA-siRNA interference or dBcAMP and 6. mu. g/ ml D4-F treatment cells 0, 1, 2 hours; 5. 6. mu.g/ ml and 100.mu. g/ l of the adenoid acid cyclase inhibitor SQ 22536 or 25mM Cdc42 inhibitor, Sramine B, incubated at 37.degree. C.for 30 minutes; 6. 6. 6. mu. g/ ml of D4-F treated cells for 0, 10, 15 and 20 minutes. The activity of Cdc42 was observed. The activity of the Cdc42 was detected by the absorbance value, and the activity of Cdc42 was detected by the detection kit of the activity of the Cdc42. The liquid scintillation counter was used to detect the cholesterol efflux in the cells, and the Western blot analysis The expression of ABCA1 protein and the phosphorylation of ABCA1 were detected by enzyme-linked immunosorbent assay, and the level of cAMP was detected by enzyme-linked immunosorbent assay. Results: The expression of PKA and PKA-siRNA down-regulated the expression of PKA protein, and the expression of PKA-siRNA and dBcAMP did not affect the effect of D4-F on the increase of ABCA1 protein; and the PKA-siRNA At the same time, SQ22536 could reverse the increase of cAMP level, PKA activity and ABCA1 phosphorylation caused by D4-F, and D4-F can influence Cdc42 activity; Sramine B affects D4-F Conclusion: The time and concentration of 1D4-F are dependent on the up-regulation of ABCA1 protein.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2332636
[Abstract]:Objective: The expression of ATP-binding cassette transporter A (ABCA1) in the 3-phosphate-binding cassette transporter A (ATP-binding cassette transporter A1, ABCA1) plays an important role in the efflux of cholesterol in the cells. Apolipoprotein A-1 mimetic peptide D4-F is a small molecular peptide containing 18 amino acid residues, and the-amphoteric helical motif is a structural basis that is important for lipid binding. At present, animal experiments show that D4-F has the effect of anti-inflammatory and anti-atherosclerosis, and in the blood of the mice fed with D4-F, the level of triglyceride and low-density lipoprotein cholesterol is lower than that of normal group. It was found that D4-F can increase the level of ABCA1 protein in cell test, but its specific mechanism is not clear. Cdc42 is a member of the RhoGTP enzyme superfamily with a molecular weight of 25KD. It was found that Cdc42 was involved in the cell liner. The outflow of alcohol. Cdc42 in fibroblasts and macrophages of patients with Tangier disease After the binding of apoA-1 and ABCA1, C can be activated dc42. The study of the experimental group has demonstrated that apoA-1 has the effect of the cAMP/ PKA signaling pathway on the expression of ABCA1 protein and its mediated cholineralization. Based on these studies, we studied the effect of D4-F on the expression of ABCA1 and its mediated cholesterol, and further observed whether Cdc42/ cAMP/ PKA was in this process. The effect of the first part D4-F on the expression of ABCA1 and its medium Effect of guided cholesterol efflux: D4-F as an analog peptide of apoA-1, and whether it affects the ABC of the THP-1 macrophage-derived foam cells The purpose of this experiment was to observe the ABCA1 table in the source foam cells of the THP-1 macrophage by D4-F. Dup and its effect on cholesterol efflux. Methods: THP-1 monocytes were induced by PMA, and then added. After the induction of oxLDL to the foam cells, 1. The different concentration (0. mu.g/ ml? 2. mu.g/ ml? 4. mu.g/ ml? 6. m g/ ml) D4-F treated cells for 24 h; 2.6. mu.g/ ml D4-F for different time (0? 6? 12? 24 h); and 5. mu.g/ ml BSA treated cells for 24 hours; 3.6. mu.g/ ml D4-F and 20.mu. g/ ml D The expression of ABCA1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. The contents of total cholesterol, free cholesterol and cholesteryl ester in the cell were determined. The results showed that D4-F did not affect the level of ABC1m RNA, and the expression of ABCA1 protein reached the peak at 6. m u.g/ ml of D4-F and D4-F. After the inhibition of protein synthesis, the D4-F could significantly slow the degradation of the ABCA1 protein.; High performance liquid chromatography to show D4-F The content of total cholesterol, free cholesterol and cholesteryl ester in cells is reduced. The concentration of D4-F can be detected by liquid scintillation. The dependence and time-dependence increase the efflux of cholesterol and its phospholipids in cells. Two-part D4-F increased the expression of ABCA1 and its mechanism to mediate the outflow of cholesterol. The effect of D4-F on the expression of ABCA1 protein and its possible mechanism to mediate the outflow of cholesterol were mainly discussed. after the THP-1 monocytes were induced to adhere by PMA, oxLDL was added to induce it to be converted into foam cells, 1. 6. mu. g/ ml of D4-F treated cells for 0, 15, 20, 25 minutes, observed PKA activity; 2. After the PKA-siRNA was transfected, 6. mu.g/ ml of D4-F was incubated for 24 hours; 3. 1. 0mM PKA specific agonist dBcAMP incubated with 6. mu.g/ ml D4-F for 24 hours; 4.20. mu.g/ ml of D4-F, and PKA-siRNA interference or dBcAMP and 6. mu. g/ ml D4-F treatment cells 0, 1, 2 hours; 5. 6. mu.g/ ml and 100.mu. g/ l of the adenoid acid cyclase inhibitor SQ 22536 or 25mM Cdc42 inhibitor, Sramine B, incubated at 37.degree. C.for 30 minutes; 6. 6. 6. mu. g/ ml of D4-F treated cells for 0, 10, 15 and 20 minutes. The activity of Cdc42 was observed. The activity of the Cdc42 was detected by the absorbance value, and the activity of Cdc42 was detected by the detection kit of the activity of the Cdc42. The liquid scintillation counter was used to detect the cholesterol efflux in the cells, and the Western blot analysis The expression of ABCA1 protein and the phosphorylation of ABCA1 were detected by enzyme-linked immunosorbent assay, and the level of cAMP was detected by enzyme-linked immunosorbent assay. Results: The expression of PKA and PKA-siRNA down-regulated the expression of PKA protein, and the expression of PKA-siRNA and dBcAMP did not affect the effect of D4-F on the increase of ABCA1 protein; and the PKA-siRNA At the same time, SQ22536 could reverse the increase of cAMP level, PKA activity and ABCA1 phosphorylation caused by D4-F, and D4-F can influence Cdc42 activity; Sramine B affects D4-F Conclusion: The time and concentration of 1D4-F are dependent on the up-regulation of ABCA1 protein.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
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