【作者】 尹苗；

【导师】 安利国； 潘杰；

【作者基本信息】 山东师范大学 ， 动物学， 2009， 博士

【摘要】 动脉粥样硬化（atherosclerosis, AS）是由多种因素和作用环节介导的慢性进行性病理过程,其受多种遗传因素和外界因素的影响,是目前威胁人类生命的主要疾病之一,是导致冠心病、心肌梗塞等心血管病的主要病理生理基础。炎症和血脂异常是AS发生的主要动因。炎症反应过程贯穿于AS发生发展的各个阶段,在内皮细胞损伤、炎症细胞浸润、泡沫细胞形成、平滑肌细胞迁移增殖到脂质条纹、纤维斑块乃至复合病变形成的过程中都有炎症因素参与,AS是慢性局部炎症的观点已被广泛认可。目前对AS的早期发病机制还不明确,往往是血脂代谢紊乱、炎症以及糖代谢失衡等多种危险因素并存,共同影响着AS的发生发展。TNF-α（tumor necrosis factor-α, TNF-α）是生物体内主要的促炎因子和免疫调节因子,参与机体的急、慢性炎症过程。在人类和小鼠的AS病变部位均可检测到TNF-α的存在。TNF-α促进血管内皮细胞粘附分子表达,刺激血管内皮细胞、成纤维细胞、单核/巨噬细胞的活化与迁移,通过诱导其分泌细胞因子等方式,发动炎症反应。但关于TNF-α在AS中,尤其是脂质条纹期的具体作用尚未被完全认识。作为一种免疫调节因子,TNF-α在AS发生过程中对其它炎症因子和细胞因子的表达具有什么影响?它在AS不同阶段中的作用是否一致?除炎症反应外,TNF-α是否还存在其它途径参与AS发生发展过程?ApoE基因缺失(apoE deficient, apoE^-/-)小鼠在普通标准饮食条件下能够自发产生AS病变,且发病过程与人类AS极为相似,是研究人类AS的理想模型,为研究人类AS的发病机理以及干预治疗提供了极大的方便。本实验利用apoE^-/-/TNF-α-/-（AT）双基因突变小鼠和apoE基因缺失(apoliprotein E deficient, apoE^-/-)小鼠为实验材料,利用血生化分析、Real-time RT-PCR、细胞因子抗体芯片等检测技术,结合冷冻切片、常规病理染色等形态学分析方法,对不同年龄条件下AT和apoE^-/-小鼠的血脂水平、主动脉AS病变进行了观察测定,同时对小鼠脾脏和主动脉中炎症相关基因,肝脏中胆固醇逆转运相关基因的转录水平进行了分析,并测定了6周龄小鼠主动脉中64种细胞因子的含量,旨在明确AS发生早期,TNF-α对apoE^-/-小鼠炎症因子等细胞因子表达的影响作用,同时进一步确定TNF-α在AS过程中可能的作用途径与作用特点。实验主要结果如下:1.小鼠主动脉根部病理形态观察和病变面积分析:病理形态观察结果表明,apoE^-/-小鼠主动脉根部内膜自4周龄起开始出现轻度的内膜脂质沉积,苏丹IV染色阳性区面积和斑块厚度随年龄增长逐渐加重。大部分AT小鼠4周龄时,主动脉根部镜检时未发现明显脂质沉积,至6周龄开始出现脂质沉积,但病变面积和病变程度要轻于同年龄段的apoE^-/-小鼠。至8周龄时,apoE^-/-和AT小鼠小鼠主动脉内膜处均出现显著脂质沉积,并伴有泡沫细胞聚集。随小鼠年龄增大,主动脉根部斑块分布区域增多,平均面积进一步增大。16周龄小鼠主动脉壁一侧部分区域苏丹IV着色不均,表明平滑肌细胞发生表型转换,迁移进入病灶部位并吞噬脂质形成平滑肌来源的泡沫细胞。至24周龄时,平滑肌细胞增殖迁移、吞噬脂质程度和病变面积进一步加重,管腔变小,血管壁变薄。病变面积分析结果表明,TNF-α促AS的作用在AS发生早期的作用更为显著。4周龄时,apoE^-/-小鼠主动脉瓣膜处平均病变面积是AT小鼠的7.81倍,6周龄时,是AT小鼠的2.58倍。随着小鼠年龄增加,TNF-α的作用有所减弱,至24周龄时,apoE^-/-小鼠主动脉根部的病变面积只比AT小鼠增加了29%。2.小鼠血脂生化指标检测:与野生型（wild type, WT）小鼠相比,apoE^-/-小鼠在各个年龄阶段均表现较为明显的脂质水平的升高。除4周龄和6周龄甘油三酯（triglyceride, TG）外,其余检测指标与WT相比均有显著性差异（P<0.05）。各年龄段TNF-α-/-小鼠的各项血脂水平与WT相比均不具有显著性差异。与apoE^-/-小鼠相比,AT小鼠的血脂水平呈现如下变化:AT小鼠各年龄段总胆固醇（total cholesterol, TC）水平均低于同年龄的apoE^-/-小鼠;AT小鼠各年龄段TG水平也均低于apoE^-/-小鼠,但自16周起才具有统计学意义（P<0.05）;apoE^-/-小鼠和AT小鼠的血浆低密度脂蛋白胆固醇（low density lipoprotein cholesterol, LDL-C）水平在各年龄段均未表现出显著性差异;4-12周龄时,血浆高密度脂蛋白胆固醇（high density lipoprotein cholesterol, HDL-C）水平apoE^-/-小鼠要高于AT小鼠,但16周龄时,AT小鼠血浆HDL-C水平已较apoE^-/-小鼠有所提高。以上结果表明,TNF-α参与了apoE^-/-小鼠脂质代谢过程,当TNF-α基因缺失后,影响了apoE^-/-小鼠的脂质代谢,而且这种影响可能是通过HDL的相关途径实现的。3.TNF-α缺失前后apoE^-/-小鼠脾脏中炎症基因转录变化特征:Real-time RT-PCR结果表明,TNF-α缺失之后,各个年龄段脾脏中炎症因子的表达均受到较大影响。与apoE^-/-小鼠相比,6周龄时,所检测的6种主要细胞因子的表达均有所下降,其中白细胞介素（interleukin , IL）10和IL-12 mRNA表达水平显著下调（P<0.05）,IL-1β和IL-4 mRNA表达水平下调极显著（P<0.01）。12周龄时,所检测的细胞因子在apoE^-/-小鼠和AT小鼠中的表达情况表达与6周时出现较大差异。与apoE^-/-小鼠相比,有4种细胞因子mRNA表达发生了显著变化:干扰素（interferon, IFN）γ和IL-1β出现了显著上调（P<0.05）,而IL-6和IL-10仍表现出显著下降的趋势（P<0.05）,而IL-4和IL-12mRNA表达水平发生轻微上调。24周龄时,所检测的各细胞因子的表达趋势与12周龄时相似,AT小鼠中IFN-γ、IL-1β、IL-4和IL-12的mRNA表达水平比apoE^-/-小鼠有所上升,而IL-6和IL-10的表达水平较apoE^-/-小鼠低,但是IL-1β、IL-4、IL-10和IL-12的差异不显著。这表明,TNF-α的缺失对于小鼠脾脏组织中炎症因子的表达具有影响作用,且这种影响作用呈现年龄依赖的特点。4.TNF-α缺失前后6周龄apoE^-/-小鼠主动脉炎症等细胞因子表达变化特征:（1）6周龄小鼠主动脉炎症基因转录变化特征:6周龄时,AT小鼠主动脉组织中IL-1β、IL-4、IL-6,IL-10、IL-12、IFN-γ的表达均较apoE^-/-小鼠有所下降,各基因表达趋势与脾脏组织基本一致,但只有IL-1β、IL-4、IL-12和IFN-γ的表达变化具有差异显著性（P<0.05）。AT小鼠主动脉中核转录因子（nuclear factor-κB, NF-κB）的转录水平显著低于apoE^-/-小鼠,而κB抑制因子（inhibitor-κB, I-κB）的表达量则较apoE^-/-小鼠有所上调。这些结果表明,TNF-α的缺失可能通过NF-κB信号途径影响了apoE^-/-小鼠主动脉组织中相关炎症因子基因转录水平。（2）6周龄小鼠主动脉细胞因子抗体芯片检测:抗体芯片检测结果表明,在所检测的64种细胞因子中,与apoE^-/-小鼠样品相比,AT小鼠主动脉样品共有20种细胞因子的表达差异超过2倍,其中2种发生上调[粘附相关激酶（adhesion-related kinase, ARK）和胰岛素样生长因子结合蛋白（insulin-like growth factor banding protein,IGFBP）-6],18种发生下调[包括细胞分化簇抗原（cluster of differentiation, CD）30、CD30 L、CD40、IL-5、IL-6、IL-10、IL-12p40/p70、KC（keratinocyte-derived chemokine）、巨噬细胞炎症蛋白（macrophage inflammatory protein, MIP）-1α、MIP-1β、MIP-1γ、MIP-3α、血小板因子（platelet factor, PF）-4、胸腺活化调节趋化因子（thymus and activation-regulated chemokine, TARC）、胸腺表达趋化因子（thymus-expressed chemokine, TECK）、基质金属蛋白酶抑制因子（tissue inhibitory of metalloproteinase, TIMP-1）、可溶性TNF受体I（soluble TNF receptor, sTNFRI）和血小板生长因子（thrombopoietin, TPO）]。表达具显著性差异的主要是趋化因子家族成员（如KC、MIP、TARC、TECK等）、受体类分子（如Ax1、CD30L、CD40和sTNFRI等）、IL类成员（如IL-5、IL-6、IL-10和IL-12p40/p70等）,另外还包括少量的调节因子（如IGFBP-6和TIMP-1）。其余细胞因子的表达差异不显著。细胞因子抗体芯片结果表明,TNF-α缺失后影响了apoE^-/-小鼠主动脉中不同类型细胞因子的表达。这些细胞因子的下调减缓了单核细胞的活化和向内皮下的迁移过程,减轻了内皮细胞与单核/巨噬细胞以及T细胞的相互作用,从而减缓了AS病变进程。5.TNF-α缺失前后apoE^-/-小鼠肝脏中胆固醇逆转运相关基因表达变化特征:TNF-α可显著影响apoE^-/-小鼠肝脏组织中胆固醇逆转运（reverse cholesterol transport, RCT）相关的基因表达,且这种影响在不同年龄阶段显现出不同的作用趋势。6周时,与apoE^-/-小鼠相比,所检测的六种基因的mRNA表达水平在AT小鼠中均发生下降,其中载脂蛋白A-I（apolipoprotein A-I, apo A-I）、肝X受体α（liver X receptor alpha, LXRα）、三磷酸腺苷结合盒A1（ATP binding cassette transporter A1, ABCA1）和β-羟基β-甲基戊二酰辅酶A还原酶（β-hydroxy-β-methyl glutaryl CoA reductase, HMG-CoAR）的表达显著下调。AT小鼠中apo A-I、卵磷脂胆固醇脂酰转移酶（lecithin-cholesterol acyltrasferase, LCAT）和B类清道夫受体I（scavenger receptor class B type I, SR-BI）的表达量自12周龄起表达量较apoE^-/-小鼠有所上调,至24周龄时、其表达量已较apoE^-/-小鼠显著上调（P<0.01）。LXRα和ABCA1表现出类似的表达特征。12周龄时,AT小鼠的表达量低于apoE^-/-小鼠,但至24周龄时,AT小鼠中的表达量也呈现出显著上调的趋势（P<0.01）。在检测的三个年龄段中,HMG-CoAR在AT小鼠中的表达量均较apoE^-/-小鼠低（P<0.05）。本实验结果说明,在apoE^-/-小鼠AS发生的不同阶段,TNF-α对胆固醇逆转运起到了不同的影响作用。6周龄时TNF-α可促进肝脏中RCT过程,而24周龄时,TNF-α又表现出较强的RCT的抑制作用,表现出其促AS活性。综上所述,TNF-α缺失可对apoE^-/-小鼠AS的发生起到一定的阻遏作用。在6周龄时,TNF-α缺失可通过下调脾脏及主动脉组织中IL-1β、IFN-γ和IL-12等I型细胞因子的转录活性,对apoE^-/-小鼠AS的进程起到抑制作用。细胞因子抗体芯片检测表明在6周龄小鼠主动脉中,AT小鼠中多种趋化因子、粘附分子MIP-1α、MIP-1β、KC、TARC、MCP-1、VCAM-1表达的下调表明,TNF-α通过影响这些成分的表达,促进了单核/巨噬细胞、T细胞、血管内皮细胞之间的相互作用,参与了泡沫细胞和脂质条纹病变的形成过程。但随着年龄增长,TNF-α对炎症因子在脾脏中的表达影响发生改变。12和24周龄时,IFN-γ、IL-1β、IL-4和IL-12的表达呈现出与6周龄相反的趋势,这提示TNF-α在apoE^-/-小鼠不同年龄阶段对炎症因子的表达具有不同的影响作用。血脂HDL-C检测结果提示,TNF-α可能通过RCT参与了apoE^-/-小鼠中脂质代谢过程。Real-time RT-PCR结果表明,小鼠肝脏中RCT相关基因的转录水平在TNF-α缺失前后发生显著变化。6周龄时,TNF-α缺失可以通过下调apo A-I、LCAT、SR-B1、LXRα和HMG-CoAR的表达抑制了RCT的进行,至24周龄时,又可以通过上调该基因的表达促进RCT。这表明TNF-α在apoE^-/-小鼠不同年龄阶段对RCT具有不同的影响作用。基于本实验研究结果,我们认为,TNF-α在apoE^-/-小鼠AS发生过程中表现出作用的复杂性和时间依赖性的特点。AS是多因素参与的复杂病变过程,TNF-α广泛参与了AS中炎症反应的调节和脂质代谢过程,并总体上表现出了促AS作用,其炎症和RCT调节表现出了不同的时间依赖性。我们推测,在AS早期病变脂质条纹时期,肝脏脂代谢紊乱可能只是一个重要始动因素,炎症在此过程中起到主导作用。更多还原

【Abstract】 Atherosclerosis （AS） is a multifactorial and progressive disease, many complicated factors and biological processes contribute to atherogenesis. The inflammatory reaction and inflammation-related factors play important roles at all stages of atherogenesis. Atherosclerosis involves a large genetic network, not a simple linear pathway. This network extends to interact with many known risk factors such as dyslipidaemia and inflammation for the disease, and involves many cell types and organ systems.Tumor necrosis factor-alpha （TNF-α）, a key inflammatory cytokine in the inflammatory cascade, is considered to plays important roles in atherogenesis. TNF-αwas produced by several types of cells, such as macrophages, vascular endothelial cells （ECs） and smooth muscle cells （SMCs）. TNF-αcould induce the production of cytokines, chemokines, and increase the expression of adhesion molecules on ECs, monocytes and leukocytes, leading to the recruitment of monocytes / macrophages and infiltration into the subendothelial space of arteries, as well as subsequent activation of T lymphocytes and the migration and proliferation of SMCs. However, its precise characters in the primary stage of the disease remain unclear. Apolipoprotein E-null (apoE^-/-) mice which spontaneously develop atherosclerosis with features similar to those observed in humans are the idea model of human atherosclerosis researches. It provides the advantage for the mechanism and intervention of atherosclerosis researches.To assess the function of TNF-αduring atherogenesis in apoE^-/- mice, a comparative study on fatty-streak lesion, serum lipid level, the mRNA level of target gene in spleen, aorta and liver of apoE^-/- and apoE/TNF-αdouble mutant （AT） mice were performed. By cytokine antibody array, 64 kinds of cytokines expression were detected in aorta with 6-week old mice. The characteristics of expression of inflammatory factors in spleen and aorta, and reverse cholesterol transport （RCT） related genes in liver were analyzed to demonstrate the role of TNF-αin atherogenesis. The results showed that:1. Pathological analysis and lesion quantificationThe results of atherosclerotic lesions in root showed that deficiency of TNF-αled to reduction of atherogenesis in apoE^-/- background. Lipid accumulation in the intima of the aorta existed as early as 4-week of age in apoE^-/- mice and were progressed with age. There was no observed lesion in the 4-week old AT mice and all ages of TNF-α-/- and WT mice. From 6-week to 24-week old, lipid deposition in the aortic sinus was most prominent in apoE^-/- mice, while it tended to be lower in AT mice. The mean lesion area revealed that the promoting effect of TNF-αon AS was more prominent in the early stage. When being 4-week old, the aorta root mean lesion area of apoE^-/- mice was 7.81 times of AT mice, but reduced to 2.58 times when at 6-week old. Furthermore, at age of 24-week old, the mean lesion area in apoE^-/- mice was only 29% larger than that of AT mice.2. Plasma lipid parametersThe serum total cholesterol （TC）, triglyceride （TG）, low density lipoprotein cholesterol ester （LDL-C） and high density lipoprotein cholesterol ester （HDL-C） levels of apoE^-/- mice were higher than that of age matched WT mice from 4-week to 24-week. Besides TG level at 4 and 6-week age, TC, TG, LDL-C and HDL-C levels in apoE^-/- mice increased significantly when compared with TNF-α-/- and WT mice （P<0.05）. The level of plasma lipid between TNF-α-/- and WT showed no significant difference at all stage. TC, TG and LDL-C levels were no statistically different between AT and apoE^-/- mice. Compared with apoE^-/- mice, the serum lipid level demonstrated that 1) TC and TG were lower in AT mice at the same age, 2) LDL levels were not shown significant differences between the two genotype at all age group. At age of 4-12 weeks, HDL levels of apoE in mice is higher than AT mice, but at 16-week, AT mouse plasma HDL-C level in AT mice increased to a level that was higher than apoE^-/- mice. These results showed that, TNF-αinvolved in the apoE^-/- mice lipid metabolism, and deletion of TNF-αgene might have impact on the lipid metabolism process through the HDL-C related pathway.3. Variation of the inflammatory factor gene transcription in spleenIn spleen, the expressions of inflammatory markers were dramatically altered in AT mice. Compared with apoE-/ - mice, the transcription level of the six cytokines were decreased, while interleukin （IL）- 1β, IL-4, IL-10 and IL-12 mRNA were significantly reduced （P<0.05） at 6-week old. At 12-week, the detection of cytokines expression between apoE^-/- and AT mice showed different pattern. Interferon-γ（IFN-γ） and IL-1βappeared significantly up-regulated （P<0.05）, IL-6 and IL-10 still showed a significant downward trend in AT mice （P<0.05）, and the IL-4 and IL-12 mRNA expression level showed a minor increase. When it came to 24- week, the trends of cytokine expression were similar with 12-weeks, IFN-γ, IL-1β, IL-4 and IL-12 transcription levels increased in AT mice, while the IL-6 and IL-10 expression level were lower than that in apoE^-/- mice, but the IL-1β, IL-4, IL-10 and IL-12 showed no significant difference between the two genotypes. These results indicated that the absence of TNF-αhad impact on the expression of inflammatory factors in spleen, and this influence showed age-dependent characteristics.4. Expression characteristics of inflammatory factors and cytokines in 6-week aorta（1） Inflammatory gene transcription in 6-week aortaAt 6-week, the expression of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γin aorta were decreased in AT mice than those in apoE^-/- mice, the gene expression pattern had the same trends with spleen, but only IL-1β, IL-4, IL-12 and IFN-γhad significant difference （P<0.05）. The transcription of nuclear factor-κB （NF-κB） level in AT mice was significantly lower, while the expression of inhibitor-κB （IκB） was higher than apoE^-/- mice. These results indicated that, the absence of TNF-αmight affect the gene transcription of inflammatory factors gene transcription by NF-κB signaling pathway.（2） Cytokine antibody array in 6-week aortaAmong the 64 cytokines detected by cytokine antibody array, expression levels of 22 cytokines were changed significantly in AT mice compared to apoE^-/- mice. Two cytokines,including adhesion-related kinase （ARK）, insulin-like growth factor banding protein （IGFBP） expression were significantly increased. Eighteen cytokines expression decreased by 2-fold or more in AT mice compared to apoE^-/- mice, including cluster of differentiation （CD）30, CD30 L, CD40, IL-5, IL-6, IL-10, IL-12p40/p70, keratinocyte-derived chemokine （KC）, macrophage inflammatory protein （MIP）-1α, MIP-1β, MIP-1γ, MIP-3α, platelet factor（PF）-4, thymus and activation-regulated chemokine （TARC）, thymus-expressed chemokine （TECK）, tissue inhibitory of metalloproteinase （TIMP-1）, soluble TNF receptor I （sTNFRI） and thrombopoietin （TPO）. The cytokines which had significant difference were mainly chemokines, receptors, ILs, and a small number of regulatory factors. The cytokine antibody array analysis showed that the deletion of TNF-αimpact cytokine expression pattern in apoE^-/- mice aorta. The reduction of cytokines slowed down the monocyte activation and migration, reduced the interaction of endothelial cells, monocytes/macrophages and T-cell, thus slowing the disease process of atherogenesis.5. Expression characteristics of reverse cholesterol transport-related gene in liverTNF-αcould significantly affect the reverse cholesterol transport-related gene expression of apoE^-/- mouse liver, and this effect showed different trends at different ages. At 6-week, the transcription level of apolipoprotein A-I （apo A-I）, liver X receptor alpha （LXRα）, ATP binding cassette transporter A1 （ABCA1）,β-hydroxy-β-methyl glutaryl CoA reductase （HMG-CoAR）, lecithin-cholesterol acyltrasferase （LCAT） and scavenger receptor class B type I （SR-BI） were decreased in AT mice. In AT mice, expression levels of apo A-I, LCAT and SR-BI started to increase from 12-week compared with apoE^-/- mice, and were significantly up-regulated at 24-week （P < 0.01）. LXRαand ABCA1 showed similar expression characteristics. The expression of HMG-CoAR in AT mice were lower than those in apoE^-/- mice all stage（P<0.05）. The experimental results showed that, at different stages of atherogenesis, TNF-αplayed different effects on the reverse cholesterol transport. TNF-αhad promoted the process of the RCT in liver at 6-week, and showed strong inhibitory effects of the RCT at 24-week.In summary, TNF-αdeficiency retarded the atherogenesis in apoE^-/- mice. At 6-weeks of age, TNF-αdeficiency inhibited the process of AS by reducing the transcription activity of IL-1β, IFN-γ, IL-12 and other type-I cytokines in spleen and aortic. Cytokine antibody microarray showed that a variety of chemokines, adhesion molecules including MIP-1α, MIP-1β, KC, TARC, MCP-1, VCAM-1 reduced in AT mice, indicated that TNF-αcould affect the expression of these components, and involved in fthe ormation of foam cells and lipid stripes by promoting the interaction between vascular endothelial cells, monocyte/macrophages and T cells. However, along with the increase of age, the effect of TNF-αon the expression of inflammatory factors in the spleen were changed. The expression of IFN-γ, IL-1β, IL-4 and IL-12 showed the opposite trend in 6-week and 24-week, suggesting that TNF-αhad different effect on the expression of inflammatory factors at different age. Lipids HDL-C level suggested that TNF-αmay adopt lipid metabolism by RCT. Real-time RT-PCR results showed that the RCT-related genes transcription level changed significantly after TNF-αdeletion. TNF-αdeficiency inhibit the RCT by down-regulated the expression of apo A-I, LCAT, SR-B1, LXRα, and HMG-CoA. To 24-week, TNF-αhad up-regulated the expression of these gene for RCT. This indicates that TNF-αhad different effects on RCT with age.Based on the present results, it was concluded that the role of TNF-αshowed complex effects and time-dependent characteristics during atherosgenesis. AS was a multi-factor involved pathology process, in which TNF-αextensively involved in the regulation of inflammatory response and lipid metabolism in AS. It was speculated that in fatty-streak period, the dyslipidemia may be just as an important initiator, while inflammation play a dominant role in this process.更多还原