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

【摘要】：動脈粥樣硬化(atherosclerosis,AS)以大、中動脈內膜形成含膽固醇、類脂質等物質的粥樣斑塊為特征。在AS形成過程中,血管內皮下巨噬細胞(macrophage)逐漸吞噬細胞外脂質并形成泡沫細胞(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AS和急性冠狀動脈綜合癥的發(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胞的激活,并進一步促進AS的發(fā)展。根據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能夠在泡沫細胞生成過程中調節(jié)一系列相關基因的表達變化。 人體內的NEFA多來源于食物中的脂類物質。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主要涉及巨噬細胞對甘油三酯(Triglyceride,TG)、膽固醇及其酯的吸收與外排,與之相關的轉錄因子和脂質代謝相關基因表達水平發(fā)生變化。PPARγ/LXR-α/ABCA1信號通路相關分子,如PPARγ、ABCA1及清道夫受體(scavenger receptor)等已經被證實能夠影響巨噬細胞內環(huán)境的穩(wěn)態(tài)。 泡沫細胞內脂滴主要以TG、膽固醇及其酯為核心,表面覆蓋單層磷脂結構,并鑲嵌著多種脂滴相關蛋白(lipid droplet associated protein)。脂滴相關蛋白對脂滴代謝及其功能發(fā)揮起到了關鍵的作用。我們實驗室既往研究已經證實了誘導細胞死亡的DFF45樣效應因子(cell death-inducing DFF45-like effector , CIDE )家族和Perilipin-ADRP-TIP47(Perilipin-ADRP-TIP47,PAT)家族成員在泡沫細胞形成過程中的表達水平變化顯著,提示其參與了AS形成的調控過程。在NEFA干預下,泡沫細胞內脂質的儲積、外排以及該過程的具體調控機制目前尚不十分清楚。 針對AS的發(fā)生發(fā)展過程,用體外細胞系模擬AS泡沫細胞形成過程,并檢測NEFA作用下細胞脂質代謝情況的變化將對進一步了解AS的發(fā)病機制提供一定的參考意義。 【目的】 1.體外培養(yǎng)人單核細胞淋巴瘤細胞(THP-1細胞)并誘導分化成泡沫細胞,模擬體內AS泡沫細胞形成過程; 2.NEFA預處理THP-1源性巨噬細胞,找出NEFA孵育巨噬細胞的最佳時間和劑量; 3.形態(tài)學觀察和測定NEFA干預下的泡沫細胞中脂滴形態(tài)及相對面積變化; 4.檢測并對比分析泡沫細胞中總膽固醇(Total cholesterol,TC)和TG的含量; 5.檢測在THP-1源性泡沫細胞形成過程中脂滴相關蛋白在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泡沫細胞形成中發(fā)揮的的具體作用和可能的機制。 【方法】 1.使用佛波酯(phorbol myristate acetate,PMA)孵育THP-1細胞48h誘導生成巨噬細胞,再加入氧化型低密度脂蛋白(oxidized-lipoprotein, ox-LDL)繼續(xù)誘導48h,最終形成泡沫細胞; 2.使用MTT法檢測在不同時間和劑量梯度下NEFA對巨噬細胞生存率的影響,繪制生存曲線; 3.油紅O染色巨噬細胞/泡沫細胞內脂滴,觀察脂滴形態(tài)并采圖,使用Image-Pro Plus 6.0軟件測量各NEFA處理組及空白對照組的細胞內脂滴相對面積; 4.使用Wako生化檢測試劑盒檢測泡沫細胞中的TG和TC含量; 5.Real-time PCR方法檢測THP-1源性巨噬細胞和泡沫細胞在NEFA作用下脂代謝轉錄因子及脂滴相關蛋白在轉錄水平的變化情況。 【結果】 1. NEFA在濃度為100μM,時間為72h的條件下能夠發(fā)揮較好的孵育效果,細胞生存狀態(tài)良好。NEFA的干預能夠使泡沫細胞中脂質含量增多,但是PUFA處理組的細胞脂質蓄積量少于PA及OA處理組,脂滴相對總面積較小,細胞內的TC含量也較少。PUFA處理組PPARγ,CD36和SR-AI水平上調,同時ABCA1水平升高。 2.脂滴相關蛋白CIDE和PAT家族成員mRNA檢測結果顯示: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等主要出現于未成熟脂滴表面的蛋白水平升高。PUFA在泡沫細胞形成過程中不僅相對降低TC的水平,并且抑制脂滴的成熟從而延緩了泡沫細胞的形成。 【結論】 PPARγ/LXR-α/ABCA1信號通路的脂代謝相關基因調節(jié)巨噬細胞對脂質的攝入和外排,同時脂滴相關蛋白表達量變化與泡沫細胞內脂滴的形態(tài)大小和脂質含量變化關系密切。NEFA能夠促進THP-1源性巨噬細胞內的脂質儲積,其中PUFA可以相對的減少細胞內TC含量,對于泡沫細胞脂質儲積有延緩作用。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

【共引文獻】

相關期刊論文 前10條

1 於惠敏,宋懷東,陳家倫;脂肪分化相關蛋白的研究進展[J];國外醫(yī)學.內分泌學分冊;2004年03期

2 余熠;谷衛(wèi);;Adipophilin——脂肪細胞早期分化的標志[J];國外醫(yī)學(內科學分冊);2005年11期

3 張春利;姚元慶;李東紅;張偉;;Altered expression of adipose differentiation-related protein gene in placental tissue of pre-eclampsia[J];Journal of Medical Colleges of PLA;2006年02期

4 郝娟;羅軍;王紫騫;趙旺生;孫雨婷;鐘瑜;李君;;西農薩能羊47kD尾部相互作用蛋白基因(TIP47)的cDNA克隆、序列結構分析與組織表達[J];農業(yè)生物技術學報;2011年05期

5 馮小路;路金才;辛海量;張磊;王玉亮;;野西瓜果實超臨界CO_2流體萃取物的化學成分及藥理活性[J];上海交通大學學報(農業(yè)科學版);2009年01期

6 袁中華,楊永宗,尹衛(wèi)東,易光輝,吳孟津;Adipophilin在動脈粥樣硬化病變和脂質負荷細胞中的作用研究(英文)[J];生物化學與生物物理進展;2003年04期

7 劉清南;戴志兵;劉志強;唐朝克;田國平;戴肖松;何求;葉玲;袁中華;;高表達和敲減adipophilin對細胞內ERK1/2活性、PPARγ表達和細胞內脂質蓄積的影響(英文)[J];生物化學與生物物理進展;2011年12期

8 王中群;袁中華;;一個新的脂質蓄積標記物——親脂蛋白[J];生命的化學;2005年06期

9 孫月娥;夏文水;;硅膠柱色譜和反相高效液相色譜結合分離制備亞油酸海藻糖單酯[J];食品與發(fā)酵工業(yè);2008年12期

10 王中群,袁中華;Adipophilin在動脈粥樣硬化脂質蓄積中的作用[J];國際病理科學與臨床雜志;2005年05期

相關會議論文 前1條

1 王中群;袁中華;;脂肪分化相關蛋白與脂質蓄積[A];第八次全國動脈硬化性疾病學術會議論文集[C];2005年

相關博士學位論文 前7條

1 陳鳳玲;糖尿病大血管病變相關基因的克隆及功能初步研究[D];復旦大學;2006年

2 吳艷;肉鴨脂肪性狀相關候選基因的克隆、表達及其與胴體和脂肪性狀的關聯分析[D];西北農林科技大學;2008年

3 何雪峰;三七皂苷影響動脈粥樣硬化泡沫細胞形成的物質基礎及其機制研究[D];第三軍醫(yī)大學;2007年

4 楊淑娟;銀杏葉提取物增強骨髓間充質干細胞治療糖尿病作用研究[D];吉林大學;2009年

5 劉洪瑜;牛脂肪代謝相關基因遺傳分析及其與秦川牛經濟性狀關聯分析[D];西北農林科技大學;2009年

6 賴巧紅;SUMO化修飾對胰島β細胞凋亡的調控其機制研究[D];華中科技大學;2013年

7 吳紅梅;上市品種“康復新液”的藥學再評價[D];成都中醫(yī)藥大學;2013年

相關碩士學位論文 前10條

1 薛玲;LSDP5對小鼠心肌細胞脂滴代謝作用的初步研究[D];第四軍醫(yī)大學;2011年

2 郝娟;西農薩能羊TIP47基因的克隆分析與RNA干擾研究[D];西北農林科技大學;2011年

3 張立杰;C/EBPα對脂滴相關蛋白LSDP5轉錄活性的調控[D];華中農業(yè)大學;2011年

4 張春利;脂代謝相關基因ADRP和LPL在妊娠高血壓綜合征胎盤組織中的表達及意義[D];第四軍醫(yī)大學;2005年

5 楊永青;EMO和RH對大鼠前體脂肪細胞增殖與分化的影響[D];西北農林科技大學;2005年

6 趙雪蓮;豬ADFP基因的克隆、染色體定位及其生物學功能研究[D];華中農業(yè)大學;2005年

7 余熠;105例住院糖尿病患者死因分析[D];浙江大學;2006年

8 王中群;蛋白激酶C活性變化對adipophilin介導脂質蓄積的影響[D];南華大學;2006年

9 賈薇;Adipophilin通過ACAT1促進THP-1巨噬細胞蓄積脂質[D];南華大學;2007年

10 黃諳非;Adipophilin促進RAW264.7細胞內ACAT1高表達[D];南華大學;2008年

，

本文編號：1749546