【作者】 鲁艳军；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 流行病学研究显示：高甘油三酯血症是冠心病(Coronary heart disease,CHD)的危险因素,在高甘油三酯血症的人群中,动脉粥样硬化(Atherosclerosis,AS)的发生与血浆中增高的极低密度脂蛋白(Very low density lipoprotein,VLDL)及VLDL来源的残余脂质颗粒成正相关。我国膳食以高糖低脂为主,在血脂水平和组分上与西方人群存在显著差异,血脂紊乱以内源性甘油三酯(Triglyceride,TG)升高最为多见。TG主要存在于富含甘油三酯脂蛋白(Triglyceride-rich lipoprotein,TRL)中,而后者90%以上为VLDL,因此TG升高实际上反映了VLDL增多。VLDL是一种主要由TG,游离和酯化胆固醇,磷脂及载脂蛋白组成的脂蛋白颗粒,直径小的VLDL能够到达血管内皮下导致巨噬细胞内脂质的堆积,从而形成泡沫细胞。巨噬源性泡沫细胞是AS斑块内出现的特征性病理细胞。由于目前大多研究仅局限于与泡沫细胞形成的调节脂质代谢的相关蛋白质,如对摄取脂质相关的受体,低密度脂蛋白受体相关受体(Low density lipoprotein receptor,LRP),低密度脂蛋白受体(Low density lipoprotein recepter LDLR),MSR,CD36等,或对其胞内堆积或外排的研究,研究多是“点对点”的研究,很少考虑巨噬细胞在吞噬脂蛋白后是否伴随其他的病理生理改变。如有报道显示,吞噬大量脂质的泡沫细胞在动脉粥样进展的后期容易发生凋亡等。因而,巨噬细胞在吞噬大量脂质后胞内必然发生的比较复杂的蛋白质组改变。蛋白质组学从整体的角度分析细胞内的蛋白质表达水平或修饰状态的变化,了解蛋白质之间相互作用与联系,在蛋白质水平上发现和探讨生命活动的规律及重要病理生理的本质。目前关于泡沫细胞的蛋白组学研究已有开展,多集中在富含胆固醇的泡沫细胞研究。然而,富含TG的泡沫细胞的蛋白质组研究还未见报道。本研究首次对富含TG的泡沫细胞进行了蛋白质组研究。首先,培养的THP-1细胞经佛波脂(Phorbol-12-myristate-13-acetate,PMA)诱导48小时分化为巨噬细胞。密度梯度离心提取脂蛋白VLDL及LDL,LDL经CuSO4氧化,定量后以50μg/ml VLDL及OX-LDL孵育巨噬细胞48小时,OX-LDL处理组作阳性对照组。油红染色显示VLDL孵育的巨噬细胞内堆积大量的脂质。进一步测定胞内脂质成分,结果提示：VLDL孵育形成的泡沫细胞内主要以TG堆积为主,表明富含TG的泡沫细胞模型已建立。下一步,提取上述富含TG的泡沫细胞的总蛋白,通过二维电泳技术分离其蛋白质组,建立了富含TG的泡沫细胞的二维图谱。对二维图谱进行ImageMaster软件分析结果显示：每张图谱可检测到1000个蛋白质点左右,大部分蛋白质的等电点集中在PH 5-7。VLDL处理组与PBS处理组的蛋白质组表达量比较分析,114个蛋白质点表达上调,101个蛋白质点表达下调。这些结果表明巨噬细胞在吞噬VLDL形成泡沫细胞中,可能有大量蛋白质表是参与其病理过程。其次,为进一步鉴定上述蛋白质组中的差异蛋白质,我们在凝胶上手工切取差异点,然后经胰蛋白酶解,将上述酶解的片断经基质辅助激光解析电离飞行时间质谱分析(Matrix assisted laser desorptionionization-time of flight-mass spectrometry,MALDI-TOF-MS)分析,获得了蛋白质肽质量指纹图谱(petide mas fingerprint,PMF),然后搜索基因组和蛋白质组数据库,实现了蛋白质的定性鉴定。其中14个差异蛋白质点通过质谱分析被鉴定,其中包括与泡沫细胞中脂滴形成密切相关的蛋白质脂肪分化相关蛋白(Adipose differentiation-related protein,ADRP),磷酸甘油酸变位酶等参与糖酵解以及参与氧化应激等蛋白质,同时我们通过Western Blot验证部分蛋白质的表达变化,其结果与蛋白质组结果一致,并通过RT-PCR检测发现上述蛋白质在mRNA水平上已发生变化。上述结果表明,巨噬细胞泡沫化的过程中,不仅大量脂质堆积,可能伴随着复杂的生物学变化如糖代谢,氧化应激的变化等。另外,本文首次将荧光差异凝胶电泳(Difference in-gel electrophoresis, DIGE)技术应用于富含TG的泡沫细胞研究。由于DIGE可以将不同的标本标记不同的荧光染料,混合后在同一次二维电泳过程中进行等电聚焦和SDS-PAGE电泳,重复性好、灵敏度高,目前已作为蛋白质组定量研究的工具之一。在前期实验基础上,本实验将提取的各组细胞总蛋白各50gg分别用Cy3,Cy5标记,各样品组的等量混合物用Cy2标记作内标,凝胶匹配率达93%,经分析表达量上调1.5倍以上有94个,表达量下调1.5倍以上有78个。与传统二维电泳相比,荧光二维差异电泳具有更高的灵敏度,更好的重复性。综上所述,本课题将二维电泳技术应用于富含TG的脂蛋白致泡沫细胞的蛋白质组研究。通过二维电泳技术建立了富含TG的泡沫细胞的二维图谱；通过Image master6.0分析软件分析了在VLDL孵育的泡沫细胞中表达上调的蛋白质点114个,表达下调的蛋白质点101个；通过质谱鉴定出14个蛋白质,包括与TG在泡沫细胞中堆积密切相关的ADRP,及糖代谢中的磷酸甘油酸变位酶,烯醇化酶等。通过对这些蛋白质功能分析,提示VLDL不仅影响泡沫细胞脂质堆积,还可能影响泡沫细胞其他生物学行为(糖代谢,活性氧的生成等)；初步将DIGE技术应用于富含TG的泡沫细胞蛋白质组学研究,建立了良好的荧光二维差异电泳图谱。更多还原

【Abstract】 It has been reported that hypertriglyceridemia is a risk factor for coronary heart disease. Atherosclerogenesis in hypertriglyceridaemic individuals is associated with the increased concentrations of VLDL and VLDL-associated remnant particles.In China, the people diet characterize low-fat and high glucose, the level and composition of lipids are different from the crowd with the West, the lipid disorders within the endogenous triglyceride (TG) increased is the most prevalent. TG is most in triglyceride-rich lipoprotein, while more than 90% the latter is VLDL, thus elevated TG in fact reflects the increase in VLDL. VLDL is a large lipoprotein particle that is composed of triglycerides, free and esterified cholesterol, phospholipids, and apolipoproteins, VLDL with the small diameter can enter vascular endothelium and cause lipid accumulation in macrophages.The macrophage-derived foam cells are the characteristics of pathological-inflammation cells occuring within the AS plaque. More traditional researchs focused on foam cell induced by cholesterol-rich lipoprotein oxidizd LDL. Because of most studies work on the proteins related to the formation of foam cells, which the function is regulating lipid metabolism, such as the receptor intaking lipid, LRP, LDLR, MSR, CD36 and so on, or related to the intracellular accumulation or efflux studies, and research studies are mostly point to point, with little regard whether the other pathophysiological changes have occured in macrophages after phagocytosis lipoprotein. It has been reported that the foam cells containing large amounts of lipids in the latter progress atherosclerotic prone to apoptosis.Thus, the more complex changes in the proteome must have occured in macrophages after the phagocytosis a large number of lipids.Proteomics point of view of the overall intracellular protein expression levels or modification of the state of change, to understand the interaction between proteins and the link,to explore the laws of life activities and its important pathophysiological nature at the protein level.At present, the proteomics research on foam cell has been undertaken, mainly concentrating in cholesterol-rich foam cell.However, the proteomics research on triglyceride-rich foam cells has not been reported.In this study, the cultured THP-1 cells were treated with phorbol ester (PMA) to induce for 48 hours, the cells were differentiated into macrophages.Extracting of LDL and VLDL by density gradient centrifugation, then 50μg/ml VLDL or oxidized-LDL after quantitation was used to incubate the differentiated macrophages for 48 hours.Oil red staining showed a large number of lipid accumulation in macrophages incubated with VLDL. Further determination of intracellular lipid components, the results suggest that the foam cells incubated with VLDL accumulate mainly in triglycerides, the triglyceride levels are 5.6-fold in contrast to the PBS-treated control macrophages.The experimental results showed that in vitro cultured macrophages can form foam cells after incubation with VLDL, and the lipid composition is major as triglycerides.Next, we established triglyceride-rich foam cells of two-dimensional map by two-dimensional electrophoresis. The results showed that over 1000 spots could be detected in each gel, the isoelectric point of most proteins was in the PH 5-7.114 protein spots were increased and 101 protein spots were down-regulated in VLDL-treated macrophage fom cells compared to the proteins in PBS-treated macrophage. Further, in order to identify the protein group, the protein spots were excised by hand, and then degraded by trypsin, the enzyme fragment were analysied by MALDI-TOF-MS to obtain protein peptide mass fingerprint (peptide mas fingerprint, PMF), then were searched in genomics and proteomics databases to achieve a qualitative identification of the proteins.Of which 14 different protein spots were identified by mass spectrometry, which includes the ADRP related to lipid droplets formation in foam cells, phosphoglycerate conjugation enzymes involved in glycolysis and the protein participating in oxidative stress.Meantime, the expression of some proteins was verified by Western Blot, and the results are consistent with the proteomic results, while their mRNA level was detection by RT-PCR, which also has changed. So in the process of formation foam cells, not only a large number of lipid accumulation in macrophage, but also accompanied other complex biological changes.Emerging fluorescence difference gel electrophoresis (difference in-gel electrophoresis, DIGE) technology can label the different specimens with different fluorescent dyes, the samples are mixed and seperated by isoelectric focusing and SDS-PAGE electrophoresis at the same time. Because of its good repeatability, high sensitivity, as it is now one of the tools of quantitative proteome research. However, the applications on their atherosclerosis have not been reported. On the basis of two-dimensional map of triglyceride-rich foam cells in previous study,50p,g of total cellular protein extracted in each group were used to be labled with Cy3 or Cy5,Cy2 as internal standard, captured the images with Typhoon 9410 scanner. Higher quality two-dimensional maps were eatablised, the matching rate of gel were 93%,94 protein spots were increased and 78 protein spots were down-regulated in VLDL-treated macrophage fom cells compared to the proteins in PBS-treated macrophage (1.5 fold).Fluorescence two-dimensional difference gel electrophoresis has a higher sensitivity, the analysis of differences in the protein are more reliable.In conclusion, the two-dimensional electrophoresis technology was used to carry out a proteome research for triglyceride-rich lipoprotein-induced foam cell in this study. A triglyceride-rich foam cells of the two-dimensional map was established by two-dimensional electrophoresis technology; 114 protein spots were increased and 101 protein spots were decreased in the foam cells incubated with VLDL with the analysis of Image master 6.0 software;14 proteins, including ADRP related to triglyceride accumulation in foam cells, Phosphoglycerate mutase, enolase related to glucose metabolism and so on, were identified with mass spectrometry. With the analysis of functions of these proteins, suggesting that VLDL not only affects the lipid accumulation in foam cells, may also affect other biological action (glucose metabolism, reactive oxygen species generation, etc.)in foam cells;Initially, the two-dimensional fluorescence difference gel electrophoresis technology were used for triglycerides-rich foam cells proteomic research, the map of two-dimensional fluorescence difference gel electrophoresis has been established.更多还原