载脂蛋白A-Ⅰ通过PKA信号途径影响ABCA1的表达与功能

以THP-1巨噬细胞源性泡沫细胞为研究对象,观察载脂蛋白A-Ⅰ与三磷酸腺苷结合盒转运体A1(ATP binding cassette transporter A1,ABCA1)的相互作用,并探讨它们相互作用的机制,以便了解载脂蛋白A-Ⅰ和ABCA1在动脉粥样硬化发生发展中的作用.THP-1巨噬细胞源性泡沫细胞经各种因素处理后,采用油红“O”染色,观察细胞内的脂滴,运用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,用逆转录-聚合酶链反应和蛋白质印迹分析法分别检测ABCA1 mRNA与ABCA1蛋白质的水平.实验结果显示,载脂蛋白A-Ⅰ和腺苷酸环化酶激动剂Forskolin(FRK)引起THP-1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯减少,而腺苷酸环化酶抑制剂SQ-22536引起THP-1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯增加.载脂蛋白A-Ⅰ引起THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平和细胞内胆固醇流出增加.FRK引起THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平和细胞内胆固醇流出呈时间和浓度依赖性增加.SQ-22536引起THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平和细胞内胆固醇流出减少.结果提示,载脂蛋白A-Ⅰ可提高THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平,增加细胞内胆固醇流出,降低细胞内胆固醇聚积.其机制可能是通过PKA信号途经使细胞ABCA1蛋白质水平增加.     

转运蛋白ABCG1/4 和ABCA1对脑胆固醇的调节作用

脑是富含胆固醇的器官,机体大约有25%的胆固醇集中在脑组织中.ATP结合盒超家族转运蛋白对脑组织中胆固醇的膜外转运和动态平衡起着重要的调节作用.研究发现,ATP结合盒超家族转运蛋白亚体ABCG1、ABCG4和ABCA1在成体脑组织中存在不同程度的表达,一种或多种亚体的缺失可以导致神经退行性病变.然而,ATP结合盒超家族转运蛋白亚体对脑发育过程中脑胆固醇动态变化的调节缺乏相关性的报道.在本研究中,从低胆固醇饮食喂养的C57BL/6J小鼠中获取出生后不同发育时期的脑组织,对ABCG1、ABCG4和ABCA1的mRNA与蛋白质表达水平进行测定,并对脑组织和血清中ATP结合盒超家族转运蛋白的表达水平与胆固醇水平的相关性进行研究.同时,使用ABCG1、ABCG4单一基因敲除鼠和ABCG1、ABCG4双基因敲除鼠,研究ATP结合盒超家族转运蛋白对与胆固醇合成的相关基因表达的影响以及对脑组织胆固醇代谢的调节作用.结果发现,ABCG1、ABCG4和ABCA1在机体多个器官中均有表达,但ABCG1和ABCG4在小鼠脑组织中表达量最高.在脑组织发育过程中,ABCG1和ABCG4mRNA水平呈现明显的表达时效性,小鼠于出生后42天达到峰值,而ABCA1 mRNA的表达水平无明显变化.血清和脑组织中中酯化型胆固醇水平呈双高峰分布,也于出生后42天达到最高.基因敲除鼠模型显示,单一敲除ABCG1或者ABCG4基因对脑组织胆固醇水平无明显影响,而ABCG1和ABCG4基因的同时缺失导致脑胆固醇水平显著升高,并明显降低胆固醇合成相关基因的表达水平.本研究表明,在脑发育成熟过程中,ATP结合盒超家族转运蛋白亚体ABCG1和ABCG4,而非ABCA1,以调节脑胆固醇的膜外转运;ABCG1和ABCG4互补调控脑胆固醇的动态平衡.     

固醇调节元件结合蛋白的研究

细胞膜中的胆固醇浓度保持动态平衡有赖于固醇调节元件结构蛋白（SREBPs)。SREBPs是DNA结合蛋白,当细胞内固醇减少时,SREBPs裂解激活蛋白（SCAP）能感受到,SCAP与SREBPs形成复合物,护送SREBPs从内质网到高尔基体,再经过两个顺序性的蛋白裂解,SREBPs的N端结构域从膜上释放出发,含有bHLH-Zip的该结构域进入到细胞核,与固醇调节元件相结合,进而激活编码胆固醇生物合成的酶和LDL受体基因的转录;当胞内固醇超载时,SCAP不再护送SREBPs到高尔基体进行蛋白裂解,核内SREBPs的N端结构域也发生降解,胆固醇合成停止。若此信号传导中任一环节有误,会导致体内固醇代谢紊乱,引起高脂血症。     

Scap-Insig-2-25HC复合物与细胞内胆固醇水平的调控

固醇调节原件结合蛋白(sterol regulatory element-binding protein,SREBP)是调节细胞内固醇类物质水平的重要细胞核转录因子,通过负反馈机制维持细胞内固醇稳态.SREBP裂解激活蛋白(SREBP-cleavage activating protein,Scap)和胰岛素诱导基因2(insulin-induced gene-2,Insig-2)、25-羟基胆固醇(25-hydroxycholesterol,25HC)对SREBP激活、成熟及核转位具有重要调节作用.最近研究揭示了Scap-Insig-2-25HC复合物的分子结构,这对细胞内胆固醇代谢研究具有重要的意义.     

MicroRNAs与脂类代谢

本文综述了miRNAs在脂类代谢调控以及脂肪酸对miRNAs影响的研究进展.miRNA能够在转录后水平参与脂类代谢的多个层面,其中miR-122、miR-370、miR33等能够通过与靶基因(Cpt1α、ABCA1等)结合,直接或间接调节细胞内脂肪酸生成、脂肪酸氧化、甘油三酯合成、胆固醇流动及脂蛋白合成等多个路径.而饮食中的脂类,尤其是必需脂肪酸,能够通过对miRNAs表达的调节参与到包括癌症抵抗、炎症缓解等多个生物学进程中.     

对氧磷降低RAW264.7巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出

三磷酸腺苷结合盒转运体A1(ABCA1)是体内胆固醇逆向转运的关键环节.对氧磷是广泛使用的有机磷农药的活性代谢产物.研究发现,对氧磷能增加巨噬细胞中胆固醇的堆积,但具体机制还不清楚.以RAW264.7巨噬细胞源性泡沫细胞为研究对象,观察对氧磷对RAW264.7巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出的影响并探讨其机制.结果显示,对氧磷以时间和剂量依赖的方式增加RAW264.7巨噬细胞源性泡沫细胞中总胆固醇、游离胆固醇和胆固醇酯水平,降低ABCA1表达和胆固醇流出,同时对氧磷降低细胞中环磷酸腺苷(cAMP)的水平及腺苷酸环化酶(AC)的活性和增加磷酸二酯酶(PDE)的活性,而cAMP的类似物双丁酰环腺苷酸(dBcAMP)能够阻断对氧磷降低ABCA1表达和部分阻断对氧磷降低胆固醇流出,对氧磷导致的cAMP水平的降低也可被AC激动剂福斯高林(Forskolin)和PDE抑制剂3-异丁基-1-甲基黄嘌呤(IBMX)所阻断.以上结果表明,对氧磷通过cAMP信号通路下调RAW264.7巨噬细胞源性泡沫细胞ABCA1的表达,降低细胞内胆固醇流出和增加细胞内胆固醇堆积.     

SCAP与胆固醇水平的调节机制

SREBP裂解激活蛋白（SREBP cleavage-activating protein,SCAP）是哺乳动物脂质合成和摄入的中心调节因素。在胆固醇代谢的反馈调节系统中,SCAP与膜结合转录因子胆固醇调节元件结合蛋白（sterol regulatory element binding proteins,SREBPs）等调节因子,共同控制一系列酶编码基因的转录过程,包括胆固醇和脂肪酸生物合成过程中所需的酶。作者介绍了SREBP的结合、分类和功能及其二步蛋白水解释放;SCAP的结合和作用机制及其在胆固醇水平调节中的作用;SCAP基因缺陷型及其胆固醇水平异常,并提出了尚待解决的问题,对SCAP的研究是胆固醇水平调节领域的一个新课题。     

LXR和ABCA1对体内胆固醇代谢的调节作用

肝外组织胆固醇返回肝脏,在肝脏通过生成胆汁酸排出,这一过程称为胆固醇逆转运。研究表明LXRs在维持体内胆固醇平衡方面起着感受器作用,通过关键靶基因转录的控制来调节胆固醇分解、储存、吸收和转运。LXR和RXR激动剂可上调巨噬细胞三磷酸腺苷结合盒转运体A1和G1(ABCAl,ABCGl)的表达,导致细胞内胆固醇流出。以LXR作为靶点的药物将为治疗高胆固醇血症和抗As提供新的希望。     

NADPH氧化酶活性不影响主动脉平滑肌细胞负荷胆固醇

NADPH氧化酶产生的活性氧促进血管平滑肌细胞的增殖和迁移,与动脉粥样硬化的发生密切相关.为了观察NADPH氧化酶的亚基p47phox对血管平滑肌细胞胆固醇代谢的影响,把p47phox基因敲除小鼠的主动脉血管平滑肌细胞与10 mg/L水溶性胆固醇共孵育72 h,然后用0.3 mg/L凝血酶处理10 min,采用免疫组织化学和油红O染色、实时定量逆转录PCR、免疫蛋白印迹、细胞内胆固醇测定等方法,观察细胞内胆固醇的改变,与平滑肌细胞、巨噬细胞、炎症反应细胞内胆固醇代谢相关蛋白的表达.结果显示,与未孵育的对照组相比,水溶性胆固醇孵育过的主动脉血管平滑肌细胞内胆固醇明显增加,差别有显著性意义:细胞内中性脂滴明显增加;α-肌动蛋白的表达下降,半乳糖凝集素3表达升高,单核细胞趋化蛋白1及血管细胞黏附分子1的表达不变;ATP结合盒转运体A1、酰基辅酶A:胆固醇酰基转移酶1及脂肪分化相关蛋白的表达增加.但是,与野生型血管平滑肌细胞相比,敲除p47phox基因并不能使所测定的指标发生变化.结果提示,负荷胆固醇后,p47phox依赖的NADPH氧化酶并不能改变血管平滑肌细胞向泡沫细胞的转变.单纯敲除p47phox基因不能改变细胞内胆固醇代谢的状态.     

动脉粥样硬化中胆固醇外流的研究进展

三磷酸腺苷结合盒转运体A1(ABCA1)、三磷酸腺苷结合盒转运体G1(ABCG1)和B族Ⅰ型清道夫受体(SR-BⅠ)介导的胆固醇外流是巨噬细胞内3条主要的胆固醇外流途径,对维持细胞内胆固醇动态平衡至关重要,其中转运体的功能及其表达的调节、胞外接受体的数量和活性等对细胞内胆固醇外流效率有重要的决定作用．最新研究发现,动脉粥样硬化(As)病变中出现的脂类蓄积、炎症、氧化应激、缺氧和胰岛素抵抗等病理情况,显著影响胆固醇转运体的表达,进而影响胆固醇外流及As的发生发展．本文主要针对As病变细胞内各胆固醇外流途径的作用及常伴随的脂类蓄积、炎症、氧化应激、缺氧和胰岛素抵抗现象,对胆固醇转运体表达调节的最新进展做一综述,以期为As治疗提供新理论依据和药物靶点,推动As治疗方法的发展．     

以芳香胺玻璃为载体的固定化的胆固醇脂酶和胆固醇氧化酶

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A physiologically based in silico kinetic model predicting plasma cholesterol concentrations in humans

Increased plasma cholesterol concentration is associated with increased risk of cardiovascular disease. This study describes the development, validation, and analysis of a physiologically based kinetic (PBK) model for the prediction of plasma cholesterol concentrations in humans. This model was directly adapted from a PBK model for mice by incorporation of the reaction catalyzed by cholesterol ester transfer protein and contained 21 biochemical reactions and eight different cholesterol pools. The model was calibrated using published data for humans and validated by comparing model predictions on plasma cholesterol levels of subjects with 10 different genetic mutations (including familial hypercholesterolemia and Smith-Lemli-Opitz syndrome) with experimental data. Average model predictions on total cholesterol were accurate within 36% of the experimental data, which was within the experimental margin. Sensitivity analysis of the model indicated that the HDL cholesterol (HDL-C) concentration was mainly dependent on hepatic transport of cholesterol to HDL, cholesterol ester transfer from HDL to non-HDL, and hepatic uptake of cholesterol from non-HDL-C. Thus, the presented PBK model is a valid tool to predict the effect of genetic mutations on cholesterol concentrations, opening the way for future studies on the effect of different drugs on cholesterol levels in various subpopulations in silico.     

高效液相色谱测定微量胆固醇氧化产物

介绍一种高效液相色谱测定胆固醇氧化产物的方法,以苯甲酰氯为衍生剂,将胆固醇及其氧化产物衍生成苯甲酸酯,反相高效液相色谱分离,紫外检测,内标法定量．本法灵敏度高,重现性好,可应用于胆固醇纯度标准物质的杂质分析及血清中的胆固醇氧化产物测定。     

A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells.     

Ezetimibe inhibits the incorporation of dietary oxidized cholesterol into lipoproteins

Oxidized cholesterol is present in significant quantities in the typical Western diet. When ingested, oxidized cholesterol is absorbed by the small intestine and incorporated into both chylomicrons and LDL, resulting in LDL that is more susceptible to further oxidation. Feeding studies in animal models and epidemiological studies in humans have suggested that oxidized cholesterol in the diet increases the development of atherosclerosis. In this study, we determined the effect of ezetimibe, a drug that inhibits small intestinal absorption of cholesterol, on the levels of oxidized cholesterol in the serum after a test meal containing oxidized cholesterol. We demonstrate that ezetimibe, 10 mg per day for 1 month, markedly reduced the levels (50% decrease) of oxidized cholesterol in the serum after feeding a test meal containing either alpha-epoxy cholesterol or 7-keto cholesterol, two of the predominant oxidized cholesterols found in the diet. Moreover, the decrease in oxidized cholesterol in the serum was attributable to a decrease in the incorporation of dietary oxidized cholesterol into both chylomicrons and LDL. Because there was no decrease in postprandial triglyceride levels, we conclude that this decrease in oxidized cholesterol levels in the serum is attributable to decreased absorption and not to enhanced clearance. Whether this decrease in oxidized cholesterol absorption prevents or delays the development of atherosclerosis remains to be determined.     

Prevalence of coronary heart disease risk factors in a Cambridge, UK study

This paper examines the distribution and prevalence of risk factors for coronary heart disease in a sample of 165 men and 202 women over 40 years of age who had earlier participated in a coronary prevention trial from a general practice in Cambridge, UK. No significant differences were observed in total cholesterol levels between men and women, and a quarter of the sample had concentrations above 6.5 mmol/l which is 250 mg/dl. There were significant sex differences in a number of risk factors with males having significantly higher prevalence of low high density lipoprotein, systolic and diastolic blood pressures, obesity, and smoking than women. About 8% of men and women were obese (as defined by a body mass index > 30), while 47% of men and 35% of women were mildly overweight (body mass index > 25). Two or more risk factors for coronary heart disease (high total cholesterol and/or hypertension and/or obesity) were present in 4% and 9% of older men and women respectively. Furthermore, about half the subjects had more than one risk factor for coronary heart disease.     

An apolipoprotein A-I mimetic dose-dependently increases the formation of prebeta1 HDL in human plasma

Prebeta1 HDL is the initial plasma acceptor of cell-derived cholesterol in reverse cholesterol transport. Recently, small amphipathic peptides composed of D-amino acids have been shown to mimic apolipoprotein A-I (apoA-I) as a precursor for HDL formation. ApoA-I mimetic peptides have been proposed to stimulate the formation of prebeta1 HDL and increase reverse cholesterol transport in apoE-null mice. The existence of a monoclonal antibody (MAb 55201) and a corresponding ELISA method that is selective for the detection of the prebeta(1) subclass of HDL provides a means of establishing a correlation between apoA-I mimetic dose and prebeta1 HDL formation in human plasma. Using this prebeta1 HDL ELISA, we demonstrate marked apoA-I mimetic dose-dependent prebeta1 HDL formation in human plasma. These results correlated with increases in band density of the plasma prebeta1 HDL, when observed by Western blotting, as a function of increased apoA-I mimetic concentration. Increased prebeta1 HDL formation was observed after as little as 1 min and was maximal within 1 h. Together, these data suggest that a high-throughput prebeta1 HDL ELISA provides a way to quantitatively measure a key component of the reverse cholesterol transport pathway in human plasma, thus providing a possible method for the identification of apoA-I mimetic molecules.