非酯化脂肪酸在THP-1源性泡沫细胞形成过程中的作用研究

发布时间：2018-04-14 13:31

本文选题：非酯化脂肪酸 + THP-1　；参考：《第四军医大学》2011年硕士论文

【摘要】：动脉粥样硬化(atherosclerosis,AS)以大、中动脉内膜形成含胆固醇、类脂质等物质的粥样斑块为特征。在AS形成过程中,血管内皮下巨噬细胞(macrophage)逐渐吞噬细胞外脂质并形成泡沫细胞(foam cell),这是AS发生发展过程中的重要组成部分。在肥胖人群中,血液中非酯化脂肪酸(non-esterified fatty acid,NEFA)含量的升高对AS和急性冠状动脉综合症的发生有着重要的影响。在健康志愿者和2型糖尿病病人血液中,NEFA的升高有助于巨噬细胞的激活,并进一步促进AS的发展。根据脂肪酸链的饱和程度分类,NEFA主要分为饱和脂肪酸(saturated fatty acid, SFA)、单不饱和脂肪酸(monounsaturated fatty acid,MUFA)和多不饱和脂肪酸(polyunsaturated fatty acid,PUFA)。NEFA能够在泡沫细胞生成过程中调节一系列相关基因的表达变化。人体内的NEFA多来源于食物中的脂类物质。最具有代表性的游离脂肪酸包括:棕榈酸(palmitic acid,PA)、油酸(oleic acid,OA)、亚油酸(linoleic acid,LA)和二十碳五烯酸(eicosapentaenoic acid,EPA)。它们富含于牛奶(PA),植物油(OA,LA)和鱼油(EPA)中。上述四种NEFA在链长和饱和度上均不相等。PA (16:0),OA (18:1n-9)和LA (18:2n-6)在人AS脂纹及斑块中含量丰富;LA和EPA(20:5n-3)为具有代表性的两种PUFA。 AS主要涉及巨噬细胞对甘油三酯(Triglyceride,TG)、胆固醇及其酯的吸收与外排,与之相关的转录因子和脂质代谢相关基因表达水平发生变化。PPARγ/LXR-α/ABCA1信号通路相关分子,如PPARγ、ABCA1及清道夫受体(scavenger receptor)等已经被证实能够影响巨噬细胞内环境的稳态。泡沫细胞内脂滴主要以TG、胆固醇及其酯为核心,表面覆盖单层磷脂结构,并镶嵌着多种脂滴相关蛋白(lipid droplet associated protein)。脂滴相关蛋白对脂滴代谢及其功能发挥起到了关键的作用。我们实验室既往研究已经证实了诱导细胞死亡的DFF45样效应因子(cell death-inducing DFF45-like effector , CIDE )家族和Perilipin-ADRP-TIP47(Perilipin-ADRP-TIP47,PAT)家族成员在泡沫细胞形成过程中的表达水平变化显著,提示其参与了AS形成的调控过程。在NEFA干预下,泡沫细胞内脂质的储积、外排以及该过程的具体调控机制目前尚不十分清楚。针对AS的发生发展过程,用体外细胞系模拟AS泡沫细胞形成过程,并检测NEFA作用下细胞脂质代谢情况的变化将对进一步了解AS的发病机制提供一定的参考意义。【目的】 1.体外培养人单核细胞淋巴瘤细胞(THP-1细胞)并诱导分化成泡沫细胞,模拟体内AS泡沫细胞形成过程; 2.NEFA预处理THP-1源性巨噬细胞,找出NEFA孵育巨噬细胞的最佳时间和剂量; 3.形态学观察和测定NEFA干预下的泡沫细胞中脂滴形态及相对面积变化; 4.检测并对比分析泡沫细胞中总胆固醇(Total cholesterol,TC)和TG的含量; 5.检测在THP-1源性泡沫细胞形成过程中脂滴相关蛋白在mRNA水平上的变化并加以分析。 6.比较分析不同类型的脂肪酸在AS泡沫细胞形成中发挥的的具体作用和可能的机制。【方法】 1.使用佛波酯(phorbol myristate acetate,PMA)孵育THP-1细胞48h诱导生成巨噬细胞,再加入氧化型低密度脂蛋白(oxidized-lipoprotein, ox-LDL)继续诱导48h,最终形成泡沫细胞; 2.使用MTT法检测在不同时间和剂量梯度下NEFA对巨噬细胞生存率的影响,绘制生存曲线; 3.油红O染色巨噬细胞/泡沫细胞内脂滴,观察脂滴形态并采图,使用Image-Pro Plus 6.0软件测量各NEFA处理组及空白对照组的细胞内脂滴相对面积; 4.使用Wako生化检测试剂盒检测泡沫细胞中的TG和TC含量; 5.Real-time PCR方法检测THP-1源性巨噬细胞和泡沫细胞在NEFA作用下脂代谢转录因子及脂滴相关蛋白在转录水平的变化情况。【结果】 1. NEFA在浓度为100μM,时间为72h的条件下能够发挥较好的孵育效果,细胞生存状态良好。NEFA的干预能够使泡沫细胞中脂质含量增多,但是PUFA处理组的细胞脂质蓄积量少于PA及OA处理组,脂滴相对总面积较小,细胞内的TC含量也较少。PUFA处理组PPARγ,CD36和SR-AI水平上调,同时ABCA1水平升高。 2.脂滴相关蛋白CIDE和PAT家族成员mRNA检测结果显示:在PUFA处理组中,perilipin和Cidec等包被于成熟脂滴表面的蛋白水平降低,TIP47和ADRP等主要出现于未成熟脂滴表面的蛋白水平升高。PUFA在泡沫细胞形成过程中不仅相对降低TC的水平,并且抑制脂滴的成熟从而延缓了泡沫细胞的形成。【结论】 PPARγ/LXR-α/ABCA1信号通路的脂代谢相关基因调节巨噬细胞对脂质的摄入和外排,同时脂滴相关蛋白表达量变化与泡沫细胞内脂滴的形态大小和脂质含量变化关系密切。NEFA能够促进THP-1源性巨噬细胞内的脂质储积,其中PUFA可以相对的减少细胞内TC含量,对于泡沫细胞脂质储积有延缓作用。
[Abstract]:Atherosclerosis (atherosclerosis, AS) in large artery intima formation of cholesterol, characterized lipid substances such as plaque in AS. During the formation of vascular endothelial cells (macrophage) gradually engulfed extracellular lipid and foam cell formation (foam cell), which is AS an important part of the development process in obesity, blood nonesterified fatty acids (non-esterified fatty, acid, NEFA) the increase of the content of AS and acute coronary syndrome has an important influence. In healthy volunteers and patients with type 2 diabetes in the blood, the increase of NEFA may contribute to activation of macrophages, and to further promote the development of AS according to the degree of saturation of fatty acid chain, NEFA is mainly divided into saturated fatty acids (saturated fatty, acid, SFA), monounsaturated fatty acids (monounsaturated fatty, acid, MUFA) and polyunsaturated fat Fatty acid (polyunsaturated fatty, acid, PUFA).NEFA can regulate expression changes in foam cell formation in the process of a series of related genes.
The body of NEFA originated from lipid free fatty acids in food. The most representative include: palmitic acid (palmitic acid, PA (oleic acid), oleic acid, linoleic acid (linoleic OA), acid, LA) and twenty carbon (eicosapentaenoic acid, EPA five). They are rich in milk (PA), vegetable oil (OA, LA) and fish oil (EPA). The four NEFA in the chain length and saturation are not equal to.PA (16:0), OA (18:1n-9) and LA (18:2n-6) is abundant in AS fatty streaks and plaque; LA and EPA (20:5n-3) for two representative PUFA.
AS is mainly involved in macrophage on triglyceride (Triglyceride, TG), and the absorption of cholesterol and its esters, transcription factor and lipid metabolism related genes associated with changes in expression levels of.PPAR alpha gamma /LXR- /ABCA1 signaling pathway related molecules, such as PPAR gamma, ABCA1 and scavenger receptor (scavenger receptor) has been shown to effect of steady macrophage environment.
Foam lipid mainly TG, cholesterol and its esters as the core, covering the surface of a phospholipid monolayer structure, and inlaid with various lipid droplet associated proteins (lipid droplet associated protein). Lipid droplet associated protein on lipid metabolism and function plays a key role. Our previous studies have proved that DFF45 like effect cytokine induced cell death (cell death-inducing, DFF45-like effector, CIDE and Perilipin-ADRP-TIP47 (Perilipin-ADRP-TIP47) family, PAT family members) formed in the process of expression level changed remarkably in foam cells, suggesting its involvement in the regulation of the formation of AS. In NEFA intervention, lipid accumulation in foam cells, and the efflux process the detailed mechanism is still unclear.
In view of the occurrence and development process of AS, we can simulate the formation process of AS foam cells with in vitro cell lines, and detect the changes of lipid metabolism in NEFA under the action of NEFA, which will provide some reference for further understanding the pathogenesis of AS.
[Objective]
1. the human monocyte lymphoma cells (THP-1 cells) were cultured in vitro and differentiated into foamy cells to simulate the formation of AS foam cells in the body.
2.NEFA pretreated THP-1 derived macrophages, and found the best time and dosage of NEFA to incubate macrophages.
3. morphological observation and determination of the morphology and relative area of lipid droplets in the foam cells under the intervention of NEFA;
4. the contents of total cholesterol (Total cholesterol, TC) and TG in foam cells were analyzed and compared.
5. the changes in the level of lipid droplet related proteins at the level of mRNA during the formation of THP-1 derived foam cells were detected and analyzed.
6. compare and analyze the specific roles and possible mechanisms of different types of fatty acids in the formation of AS foam cells.
[method]
1., phorbol myristate acetate (PMA) was used to incubate THP-1 cells 48h to induce macrophages, then oxidized low density lipoprotein (oxidized-lipoprotein, ox-LDL) to induce 48h, and finally form foam cells.
2. the effect of NEFA on the survival rate of macrophage at different time and dose gradient was detected by MTT method, and the survival curve was plotted.
3. oil red O was used to stain lipid droplets in macrophages / foam cells. Lipid droplet morphology and mapping were observed. The relative area of lipid droplets in each NEFA treated group and blank control group was measured by Image-Pro Plus 6 software.
4. the content of TG and TC in foam cells was detected by Wako biochemical test kit.
5.Real-time PCR method was used to detect the transcriptional level of lipid metabolism transcription factors and lipid droplet related proteins in THP-1 derived macrophages and foam cells under NEFA.
[results]
1. NEFA at the concentration of 100 M, time can play a better effect of 72h incubation conditions, cell survival in good condition.NEFA intervention can make lipid content in foam cells increased, but PUFA treatment group the cellular lipid accumulation was less than PA and OA treatment group, lipid droplets is relatively smaller, the content of TC cells are less.PUFA treatment group PPAR gamma, CD36 and SR-AI levels increased, also increased the level of ABCA1.
The detection results of droplet associated protein CIDE and PAT family members mRNA 2. fat showed: in PUFA treatment group, perilipin and Cidec was coated on the surface of lipid droplets in the mature protein level decreased, increased protein levels of TIP47 and ADRP occurred mainly in the surface of the lipid droplets in immature.PUFA foam cells forming process not only decreases TC the level and inhibit the maturation of lipid droplets so as to retard the formation of foam cells.
[Conclusion]
Lipid metabolism related genes of PPAR gamma /LXR- alpha /ABCA1 signaling pathway in the regulation of macrophage on lipid intake and discharge at the same time, the expression of lipid droplet associated protein changes and foam intracellular lipid droplets related to changes in morphology and lipid content closely.NEFA can promote lipid storage THP-1 derived macrophages, which can reduce the relative PUFA TC the contents of intracellular delay for foam cell lipid accumulation.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

【共引文献】