本文選題：非酯化脂肪酸 + THP-1�。� 參考：《第四軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：動(dòng)脈粥樣硬化(atherosclerosis,AS)以大、中動(dòng)脈內(nèi)膜形成含膽固醇、類脂質(zhì)等物質(zhì)的粥樣斑塊為特征。在AS形成過程中,血管內(nèi)皮下巨噬細(xì)胞(macrophage)逐漸吞噬細(xì)胞外脂質(zhì)并形成泡沫細(xì)胞(foam cell),這是AS發(fā)生發(fā)展過程中的重要組成部分。在肥胖人群中,血液中非酯化脂肪酸(non-esterified fatty acid,NEFA)含量的升高對(duì)AS和急性冠狀動(dòng)脈綜合癥的發(fā)生有著重要的影響。在健康志愿者和2型糖尿病病人血液中,NEFA的升高有助于巨噬細(xì)胞的激活,并進(jìn)一步促進(jìn)AS的發(fā)展。根據(jù)脂肪酸鏈的飽和程度分類,NEFA主要分為飽和脂肪酸(saturated fatty acid, SFA)、單不飽和脂肪酸(monounsaturated fatty acid,MUFA)和多不飽和脂肪酸(polyunsaturated fatty acid,PUFA)。NEFA能夠在泡沫細(xì)胞生成過程中調(diào)節(jié)一系列相關(guān)基因的表達(dá)變化。 人體內(nèi)的NEFA多來源于食物中的脂類物質(zhì)。最具有代表性的游離脂肪酸包括:棕櫚酸(palmitic acid,PA)、油酸(oleic acid,OA)、亞油酸(linoleic acid,LA)和二十碳五烯酸(eicosapentaenoic acid,EPA)。它們富含于牛奶(PA),植物油(OA,LA)和魚油(EPA)中。上述四種NEFA在鏈長和飽和度上均不相等。PA (16:0),OA (18:1n-9)和LA (18:2n-6)在人AS脂紋及斑塊中含量豐富;LA和EPA(20:5n-3)為具有代表性的兩種PUFA。 AS主要涉及巨噬細(xì)胞對(duì)甘油三酯(Triglyceride,TG)、膽固醇及其酯的吸收與外排,與之相關(guān)的轉(zhuǎn)錄因子和脂質(zhì)代謝相關(guān)基因表達(dá)水平發(fā)生變化。PPARγ/LXR-α/ABCA1信號(hào)通路相關(guān)分子,如PPARγ、ABCA1及清道夫受體(scavenger receptor)等已經(jīng)被證實(shí)能夠影響巨噬細(xì)胞內(nèi)環(huán)境的穩(wěn)態(tài)。 泡沫細(xì)胞內(nèi)脂滴主要以TG、膽固醇及其酯為核心,表面覆蓋單層磷脂結(jié)構(gòu),并鑲嵌著多種脂滴相關(guān)蛋白(lipid droplet associated protein)。脂滴相關(guān)蛋白對(duì)脂滴代謝及其功能發(fā)揮起到了關(guān)鍵的作用。我們實(shí)驗(yàn)室既往研究已經(jīng)證實(shí)了誘導(dǎo)細(xì)胞死亡的DFF45樣效應(yīng)因子(cell death-inducing DFF45-like effector , CIDE )家族和Perilipin-ADRP-TIP47(Perilipin-ADRP-TIP47,PAT)家族成員在泡沫細(xì)胞形成過程中的表達(dá)水平變化顯著,提示其參與了AS形成的調(diào)控過程。在NEFA干預(yù)下,泡沫細(xì)胞內(nèi)脂質(zhì)的儲(chǔ)積、外排以及該過程的具體調(diào)控機(jī)制目前尚不十分清楚。 針對(duì)AS的發(fā)生發(fā)展過程,用體外細(xì)胞系模擬AS泡沫細(xì)胞形成過程,并檢測(cè)NEFA作用下細(xì)胞脂質(zhì)代謝情況的變化將對(duì)進(jìn)一步了解AS的發(fā)病機(jī)制提供一定的參考意義。 【目的】 1.體外培養(yǎng)人單核細(xì)胞淋巴瘤細(xì)胞(THP-1細(xì)胞)并誘導(dǎo)分化成泡沫細(xì)胞,模擬體內(nèi)AS泡沫細(xì)胞形成過程; 2.NEFA預(yù)處理THP-1源性巨噬細(xì)胞,找出NEFA孵育巨噬細(xì)胞的最佳時(shí)間和劑量; 3.形態(tài)學(xué)觀察和測(cè)定NEFA干預(yù)下的泡沫細(xì)胞中脂滴形態(tài)及相對(duì)面積變化; 4.檢測(cè)并對(duì)比分析泡沫細(xì)胞中總膽固醇(Total cholesterol,TC)和TG的含量; 5.檢測(cè)在THP-1源性泡沫細(xì)胞形成過程中脂滴相關(guān)蛋白在mRNA水平上的變化并加以分析。 6.比較分析不同類型的脂肪酸在AS泡沫細(xì)胞形成中發(fā)揮的的具體作用和可能的機(jī)制。 【方法】 1.使用佛波酯(phorbol myristate acetate,PMA)孵育THP-1細(xì)胞48h誘導(dǎo)生成巨噬細(xì)胞,再加入氧化型低密度脂蛋白(oxidized-lipoprotein, ox-LDL)繼續(xù)誘導(dǎo)48h,最終形成泡沫細(xì)胞; 2.使用MTT法檢測(cè)在不同時(shí)間和劑量梯度下NEFA對(duì)巨噬細(xì)胞生存率的影響,繪制生存曲線; 3.油紅O染色巨噬細(xì)胞/泡沫細(xì)胞內(nèi)脂滴,觀察脂滴形態(tài)并采圖,使用Image-Pro Plus 6.0軟件測(cè)量各NEFA處理組及空白對(duì)照組的細(xì)胞內(nèi)脂滴相對(duì)面積; 4.使用Wako生化檢測(cè)試劑盒檢測(cè)泡沫細(xì)胞中的TG和TC含量; 5.Real-time PCR方法檢測(cè)THP-1源性巨噬細(xì)胞和泡沫細(xì)胞在NEFA作用下脂代謝轉(zhuǎn)錄因子及脂滴相關(guān)蛋白在轉(zhuǎn)錄水平的變化情況。 【結(jié)果】 1. NEFA在濃度為100μM,時(shí)間為72h的條件下能夠發(fā)揮較好的孵育效果,細(xì)胞生存狀態(tài)良好。NEFA的干預(yù)能夠使泡沫細(xì)胞中脂質(zhì)含量增多,但是PUFA處理組的細(xì)胞脂質(zhì)蓄積量少于PA及OA處理組,脂滴相對(duì)總面積較小,細(xì)胞內(nèi)的TC含量也較少。PUFA處理組PPARγ,CD36和SR-AI水平上調(diào),同時(shí)ABCA1水平升高。 2.脂滴相關(guān)蛋白CIDE和PAT家族成員mRNA檢測(cè)結(jié)果顯示:在PUFA處理組中,perilipin和Cidec等包被于成熟脂滴表面的蛋白水平降低,TIP47和ADRP等主要出現(xiàn)于未成熟脂滴表面的蛋白水平升高。PUFA在泡沫細(xì)胞形成過程中不僅相對(duì)降低TC的水平,并且抑制脂滴的成熟從而延緩了泡沫細(xì)胞的形成。 【結(jié)論】 PPARγ/LXR-α/ABCA1信號(hào)通路的脂代謝相關(guān)基因調(diào)節(jié)巨噬細(xì)胞對(duì)脂質(zhì)的攝入和外排,同時(shí)脂滴相關(guān)蛋白表達(dá)量變化與泡沫細(xì)胞內(nèi)脂滴的形態(tài)大小和脂質(zhì)含量變化關(guān)系密切。NEFA能夠促進(jìn)THP-1源性巨噬細(xì)胞內(nèi)的脂質(zhì)儲(chǔ)積,其中PUFA可以相對(duì)的減少細(xì)胞內(nèi)TC含量,對(duì)于泡沫細(xì)胞脂質(zhì)儲(chǔ)積有延緩作用。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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