【作者】 杨启红；

【导师】 司良毅；

【作者基本信息】 第三军医大学 ， 老年医学， 2008， 博士

【摘要】 糖尿病(Diabetes Mellitus,DM)患者容易发生动脉粥样硬化(atherosclerosis,AS),且时间早,进展快、范围广、预后差,即所谓加速性AS,是DM致死、致残的重要原因。研究表明,持久的高血糖状态可导致DM患者体内许多结构和功能蛋白质、脂质甚至核酸发生糖基化,经过一系列非酶促反应,即Maillard反应,形成Schiff碱,经Amadori反应重排后,最终形成具有高度活性、结构多样的糖基化终产物(advanced glycation end products,AGEs),后者通过与细胞表面特异性AGEs受体(receptor for AGEs,RAGE)相互作用而发挥一系列的血管病理作用,加速糖尿病性AS的发生、发展。泡沫细胞形成是AS发生的早期事件和关键环节,胆固醇酯在巨噬细胞中大量沉积则是泡沫细胞形成的重要原因,这一过程受清道夫受体、酰基辅酶A:胆固醇酰基转移酶-1(acyl-coenzyme A: cholesterolcyl transferase-1,ACAT-1)和ATP结合盒转运子(ATP-binding cassette transporter A1,ABCA1)共同调节。ACAT-1是细胞内唯一催化游离胆固醇和长链脂肪酸酯化形成胆固醇酯的酶,在单核/巨噬细胞中主要以ACAT-1形式存在。病理条件下,巨噬细胞ACAT-1高表达,导致细胞内胆固醇酯大量堆积而形成泡沫细胞,这就是AS的早期病变,可见ACAT-1是巨噬细胞源性泡沫细胞形成的主要介导者。然而,有关AGEs促发巨噬细胞源性泡沫细胞形成的作用是否与巨噬细胞ACAT-1的表达变化有关,目前尚不清楚。过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor-γ, PPARγ)是一类由配体活化的核转录因子,属于核受体超家族成员,与脂肪细胞分化、炎症反应、胰岛素敏感性、细胞周期调节、AS及肿瘤等关系密切。研究表明,PPARγ在单核细胞分化为巨噬细胞时表达,在维持巨噬细胞脂质代谢平衡过程中占有主导地位,但目前对于核受体PPARγ在加速性AS发生、发展中的作用尚有争议。此外,核受体PPARγ是否调节着巨噬细胞ACAT-1的转录尚未见报道。目的本研究试图从AGEs影响巨噬细胞ACAT-1和PPARγ及其下游基因表达这一角度,阐明AGEs促发AS巨噬细胞源性泡沫细胞形成的作用机制。为此,我们以体外培养的THP-1细胞为实验对象,进行以下研究:1、AGEs对巨噬细胞ACAT-1基因表达的影响; 2、AGEs对巨噬细胞核受体PPARγ及其下游SR-A基因表达的影响;3、研究比较巨噬细胞ACAT-1与PPARγ之间的相关性;4、通过观察PPARγ的合成型配体罗格列酮对AGEs诱导的巨噬细胞ACAT-1表达的作用,探讨PPARγ活化在糖尿病性AS中发挥血管保护作用可能的机制。方法1.按标准的方法用D-葡萄糖和牛血清白蛋白(BSA)共同孵育12周制备糖基化牛血清白蛋白(AGE-BSA),即AGEs。2. THP-1单核细胞培养于RPMI1640完全培养液中,于37℃、5%CO2培养箱中静置培养,每隔3～5天传代一次。用终浓度为0.1μM的PMA诱导THP-1单核细胞72h,使其分化成巨噬细胞。3.分别用浓度为50、100、200和400mg/L的AGE-BSA干预巨噬细胞24h,以100mg/L BSA作为对照组。同时用200mg/L AGE-BSA分别处理细胞0、12、24、36h和48h。4.运用免疫细胞化学方法检测巨噬细胞PPARγ及其下游SR-A的表达,了解AGEs对巨噬细胞PPARγ和SR-A蛋白表达的影响。5.运用RT-PCR和Western blot方法检测巨噬细胞ACAT-1和PPARγ基因和蛋白的表达,同使用EMSA检测巨噬细胞PPARγ的DNA结合活性,了解AGEs对巨噬细胞ACAT-1和PPARγ表达的影响。6.分别以PPARγ激动剂和PPARγ抗体刺激巨噬细胞,运用RT-PCR和Western blot方法检测巨噬细胞PPARγ和ACAT-1基因和蛋白的表达情况,明确巨噬细胞ACAT-1和PPARγ之间的相关性。7.以PPARγ激动剂罗格列酮预处理巨噬细胞4h,再用AGE-BSA刺激巨噬细胞,运用RT-PCR和Western blot方法检测巨噬细胞ACAT-1基因和蛋白的表达,明确罗格列酮对AGEs诱导的巨噬细胞ACAT-1表达的影响。结果1.荧光分光光度计上分别测定AGE-BSA和BSA的荧光值,AGE-BSA为180.5荧光单位/mg蛋白,BSA为2.98荧光单位/mg蛋白。2.经0.1μmol/L PMA诱导72h后,THP-1细胞形态由圆形变为梭形、椭圆形或不规则型,由悬浮状态向贴壁状态转化,细胞体积增大,呈阿米巴样贴壁生长,伸出伪足,富含颗粒,具有巨噬细胞的特点,转化率超过85%。3.与对照组相比,AGE-BSA可使巨噬细胞ACAT-1 mRNA和蛋白的表达水平显著上调(P<0.05),而核受体PPARγmRNA和蛋白的表达水平则显著降低(P<0.05),二者均呈现良好的浓度和时间依赖性。此外,EMSA结果显示,AGE-BSA还可浓度依赖性地抑制巨噬细胞PPARγ的DNA结合活性(P<0.05)。4.免疫细胞化学法结果显示,AGE-BSA显著抑制巨噬细胞PPARγ表达的同时,还可显著上调SR-A蛋白的表达水平(P<0.05)。5.与对照组相比,PPARγ激动剂刺激后,巨噬细胞PPARγmRNA和蛋白的表达量明显增加,而ACAT-1 mRNA和蛋白的表达量则明显降低(P<0.05);相反,PPARγ抗体刺激后,巨噬细胞PPARγmRNA和蛋白的表达量显著降低,而ACAT-1 mRNA和蛋白的表达量则显著增加(P<0.05)。6.给予1μM、5μM和10μM的罗格列酮预处理后,AGE-BSA诱导的巨噬细胞ACAT-1 mRNA和蛋白的表达量均较对照组显著下降(P<0.05)。结论1. AGE-BSA可明显增强巨噬细胞ACAT-1 mRNA和蛋白的表达水平,呈浓度和时间依赖性。2. AGE-BSA不仅可显著下调巨噬细胞核受体PPARγmRNA和蛋白的表达水平,还可浓度依赖性地抑制PPARγ的DNA结合活性。3. AGE-BSA通过上调巨噬细胞表面SR-A蛋白的表达水平,促进巨噬细胞摄取脂质,有利于泡沫细胞形成。4. PPARγ配体可使巨噬细胞PPARγ表达增强,ACAT-1表达减弱;而PPARγ抗体则使PPARγ表达减弱,ACAT-1表达增强,说明PPARγ与ACAT-1之间存在明显的负相关性。5. PPARγ特异性激动剂--罗格列酮可显著抑制AGE-BSA诱导的巨噬细胞ACAT-1 mRNA和蛋白的表达水平,此效应在一定浓度范围内呈浓度依赖性,表明PPARγ活化可通过转录调节ACAT-1的表达,从而阻止胆固醇酯在巨噬细胞内大量蓄积,最终抑制AGEs促发的巨噬细胞源性泡沫细胞形成。更多还原

【Abstract】 Diabetes mellitus is a comman, frequently-occurring disease and its chronic vascular complications such as atherosclerosis are the principal cause of morbidity and mortality in diabetic patients. It has been showed that advanced glycation end products (AGEs) play an important role in the pathogenesis and progression of accelerated atherosclerosis. AGEs are a group of nonreversible and poisonous products formed by nonenzymatic glycation of glucose with protein and lipids under hyperglycemia condition. It not only directly results in the structure and fuction changes of the tissues in which AGEs deposited but also can interact with receptor for AGEs (RAGE), a member of the immunoglobulin superfamily of cell surface molecules which express in range of cells including endothelial cell, smooth muscle cell and mononuclear phagocytes, to result in dysfunction and lesion of the cells and play a key role in initiating vasculopathy.Atherosclerosis is an inflammatory disease process with increased plasma low density lipoprotein-cholesterol level. Monocyte/macrophages contribute to the development of lesions by accumulation of cholesterol esters and the formation of foam cells. As we know, macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor (cholesterol influx), acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1; storage cholesterol ester converted from free cholesterol), and ATP-binding cassette transporter A1 (cholesterol efflux). ACAT catalyzes the formation of cholesteryl esters from cholesterol and long chain fatty acyl coenzyme A. ACAT is believed to play significant roles in lipoprotein assembly and in dietary cholesterol absorption. ACAT-1 and ACAT-2 are two subtypes of ACAT gene family. ACAT-1 protein is present at high levels in macrophages and hormone-producing cells. ACAT-1 gene expression is up-regulated in human monocytes during differentiation and foam cell formation. Under pathological conditions, accumulation of cholesteryl esters produced by ACAT-l can promote foam cell formation in atherosclerotic lesions. However, it’s still unknown the molecular mechanism of ACAT-1 gene expression, regulation and its signal transduction induced by AGEs in macrophages.Peroxisome proliferator-activated receptor-γ(PPARγ) is a member of nuclear transcription factors and involes in multiple physiological or pathophysiological process including adipocyte differentiation, inflammation, insulin sensitivity, cell cycle regulation and cancers. It is widely accepted that PPARγis a key regulater during the development of atherosclerosis. The interests have focused on the role of PPARγin modulating macrophage cholesterol and lipid homeostasis.Objectives:In this study, THP-1 macrophages were exposed to AGEs-modified bovine serum albumin (AGE-BSA) at different concentration for 24 hours and at a concentration of 200mg/L for different time to investigate the effects of AGEs on the expression of ACAT-1, PPARγand SR-A, which is in favour of clarifying the mechanism of AGEs promoting macrophage-derived foam cell formation. In additional, we investigate the effects of rosiglitazone, a synthetic PPARγligand, on the expression of ACAT-1 induced by AGEs in THP-1 macrophages. The molecular mechanisms of AGEs accelerating atherosclerosis in diabetic patients were proposed and the protective effects of PPARγactivation during foam formation were discussed.Methods:1. AGE-BSA was prepared by the methods as described previously. Briefly, AGE-BSA was made by incubating BSA with 50 mM D-glucose in 5% CO2/95% air at 37°C for 12 wk in a 10 mM PBS, pH 7.4, in the presence of 100 units/ml penicillin, and 100 mg/ml streptomycin. Unincorporated glucose was removed by dialysis overnight against 1×PBS.2. The human monocytic leukemia cell line, THP-1 cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. In order to induce phagocytic differentiation, THP-1 cells were cultured in the presence of 0.1μM PMA for 72 h.3. THP-1 macrophages were exposed to AGE-BSA at different concentrations (50, 100, 200 and 400 mg/L) for 24h, and exposed to AGE-BSA at a concentration of 200mg/L for 0,12,24,36 h and 48 h.4. Immunocytochemistry was used to examine the expression levels of proteins for PPARγand SR-A in AGEs-treated macrophages.5. The expression levels of mRNA and proteins for ACAT-1 and PPARγin AGEs-treated macrophages were semi-quantified by RT-PCR and Western blot, respectively. And the transcriptional activity of PPARγin AGEs-treated macrophages was determined by electrophoretic mobility shift assay (EMSA).6. THP-1 macrophages were incubated respectively with PPARγagonist rosiglitazone and anti-PPARγmonoclonal antibody. The expression levels of mRNA and proteins for ACAT-1 and PPARγwere detected by RT-PCR and Western blot.7. THP-1 macrophages were pretreated by rosiglitazone for 4h and then incubated with AGE-BSA at a concentration of 200mg/L for 24h. The expression levels of mRNA and proteins for ACAT-1 were detected by RT-PCR and Western blot.Results:1. AGEs-protein specific fluorescence determinations were performed by measuring emission at 450nm on excitation at 390nm using a Fluorescence spectrophotometer. AGE-BSA contained 180.5 fluorescence U/mg protein while unmodified BSA contained 2.9 fluorescence U/mg protein.2. About 85% of THP-1 cells incubated with PMA (0.1μM) for 72h stopped proliferation and differentiated into macrophages. These macrophages containing granules grew on the wall of culture flask just like ameba cells and extended pseudopods.3. Compared with the control BSA group, AGE-BSA significantly upregulated the levels of mRNA and proteins for ACAT-1 but downregulated those for PPARγin a concentration- and time-dependent manner (P<0.05). Moreover, EMSA showed that AGE-BSA also inhibited the transcription activity of PPARγin a concentration-dependent manner (P <0.05).4. Immunocytochemistry showed that AGE-BSA decreased the expression level of proteins for PPARγbut increased that of SR-A significantly (P <0.05).5. Compared with the control group, the expression of PPARγmRNA increased after PPARγagonist treated but decreased after anti-PPARγmonoclonal antibody treated (P<0.05). Moreover, the expression of ACAT-1 mRNA decreased after PPARγagonist stimulating but increased after anti-PPARγmonoclonal antibody stimulating (P<0.05). The same changes can be observed from Western blot analysis.6. Macrophages were pretreated with rosiglitazone at different concentrations (1μM, 5μM and 10μM) for 4h before exposed to 200mg/L AGE-BSA. We found that rosiglitazone impaired ACAT-1 expression induced by AGEs in a concentration-dependent manner significantly (P <0.05).Conclusions:1. AGE-BSA increased significantly the expression levels of mRNA and proteins for ACAT-1 in a concentration-and time-dependent manner.2. AGE-BSA not only decreased the expression levels of mRNA and proteins for PPARγ, but also inhibited the transcription activity of PPARγin macrophages in a concentration-dependent manner, significantly.3. AGE-BSA could promote macrophage intaking lipids by upregulating the expression of SR-A, which contributes to macrophage-derived foam cell formation.4. The increased expression of PPARγand reduced of ACAT-1 after PPARγligand stimulation suggest that PPARγmay inhibit the expression of ACAT-1.5. Rosiglitazone, a PPARγspecific agonist, could significantly inhibit the expression levels of mRNA and proteins for ACAT-1 induced by AGEs in a concentration-dependent manner, which suggests that activation of PPARγcould regulate ACAT-1 transcriptionally to prevent cholesterol esters accumulate in macrophage, thereby inhibiting the macrophage-derived foam cell formation induced by AGEs.更多还原