【作者】 王飞；

【导师】 杨笛；

【作者基本信息】 南京医科大学 ， 内科学， 2009， 硕士

【摘要】 背景动脉粥样硬化斑块的发生、发展,斑块由稳定状态演变成不稳定及破裂,涉及到脂质代谢异常、炎症反应、内皮功能受损、遗传等众多因素,其具体机制尚待阐明。共刺激分子OX40配体(OX40L/CD134L/gp34),是参与T细胞活化的辅助性分子,是肿瘤坏死因子(TNF)家族成员之一。OX40L主要表达在活化的抗原递呈细胞表面,如树突状细胞、B淋巴细胞、巨噬细胞,以及血管内皮细胞中。逆向信号通路是共刺激分子的重要特征,即除了配体/受体的正向信号,受体也可通过配体传递逆向信号。在斑块部位OX40L表达增高,OX40与OX40L的结合可诱导人脐静脉内皮细胞c-jun基因表达上调、趋化因子RANTES/CCL5表达增高,直接介导T细胞或OX40+T细胞向内皮细胞黏附。研究发现C57BL/6小鼠对动脉粥样硬化具有易感性,而C3H/ He和BALB / c的小鼠对动脉粥样硬化不易感,经过数量性状座位筛查出第一个动脉粥样硬化的易感基因区域, OX40L基因就位于该区域。这些研究显示OX40L在动脉粥样硬化的形成和发展中可能起到重要作用。内皮功能受损不仅是斑块发生的始动环节,而且加速斑块生长、解除内皮对高危斑块的保护,内皮功能的严重受损是斑块急性炎症反应的诱发因素之一。C反应蛋白(CRP)是动脉粥样硬化的炎症标志物之一,参与动脉粥样硬化的致炎过程。本实验室前期研究显示CRP可引起心肌细胞损伤,循环中受炎症因子刺激升高的CRP是否影响到内皮细胞OX40L的表达和信号通路,尚待研究。目的本论文通过观察动脉粥样硬化易感小鼠C57BL/6和不易感小鼠BALB/c心脏、肾脏、脑、骨骼肌以及脾脏等组织中OX40L基因和蛋白表达差异,CRP对小鼠主动脉内皮细胞OX40L表达的作用,探讨OX40L在动脉粥样硬化斑块形成和发展中的可能作用机制。内容与方法1. 8周龄正常雄性C57BL/6小鼠和BALB/c小鼠各8只,平均体重22克。提取小鼠心脏、脑、肾脏、骨骼肌和脾脏总mRNA和总蛋白,采用RT-PCR和Western Blot的方法分别检测OX40L的mRNA和蛋白表达水平。2.应用亲和层析柱从感染、炎症期及肿瘤患者胸水中分离、纯化CRP,电泳和Western Blot鉴定。3.采用酶消化培养法原代培养小鼠主动脉内皮细胞,用内皮细胞特异性抗体VWF抗体鉴别小鼠主动脉内皮细胞。待小鼠主动脉内皮细胞密度长至60～70%时,用100ug/ml CRP干预内皮细胞,48小时后提取内皮细胞总mRNA和总蛋白,用RT-PCR和Western Blot方法检测内皮细胞OX40L和蛋白表达水平,重复3次。结果1. C57BL/6小鼠的心脏OX40L的mRNA表达水平显著高于BALB/c小鼠(0.79±0.10 vs 0.60±0.05, P <0.05,n=8);脑、肾脏和骨骼肌mRNA表达水平在两个品系小鼠间无明显差异;BALB/c小鼠脾脏OX40L的mRNA表达显著高于C57BL/6小鼠(0.39±0.17 vs 0.25±0.04, P <0.05,n=8);C57BL/6小鼠的心脏、脑和肾脏OX40L蛋白表达水平表达较BALB/c小鼠显著增高(0.56±0.01 vs 0.19±0.07,0.18±0.07 vs 0.01±0.01,0.31±0.13 vs 0.03±0.01,P<0.05,n=8),骨骼肌和脾脏OX40L蛋白的表达水平两种品系小鼠之间无显著差异。2.纯化的CRP电泳分析纯度达95%以上,Western Blot显示纯化的CRP与CRP抗体特异性结合。3.用纯化的100ug/ml的CRP与小鼠主动脉内皮细胞共孵育48小时后,干预的内皮细胞OX40L的mRNA和蛋白质表达较未干预细胞显著增高(0.09±0.04 vs 0.76±0.12,P<0.05;0.07±0.02 vs 0.21±0.06,P<0.05,n=3)。结论OX40L在正常动脉粥样硬化易感C57BL/6小鼠和不易感BALB/c小鼠的心脏、脑、肾脏、骨骼肌和脾脏组织中基因和蛋白表达水平存在差异。CRP可诱导小鼠主动脉内皮细胞OX40L表达增高,提示OX40L可能在动脉粥样硬化中发挥作用。更多还原

【Abstract】 IntroductionThe occurrence, development and the process from stability to rapture of atherosclerotic plaques are associated with number of factors such as the disorder of lipid metabolism, involvement of immune system, endothelial dysfunction and genetic background. The exact mechanism is still under investigation. OX40 Ligand (OX40L/CD134L/gp34), a kind of co-stimulator molecules, belongs to the TNF super family. It acts as the auxiliary factor involving the process of inflammation. OX40L is mainly expressed on surface of the antigen presented cells, vascular endothelial cells and smooth muscle cells. The reverse signal pathway is an important factor of co-stimulatory molecules. It means that OX40L can also transducer the signal from its receptor, OX40L, to intracellular pathway. OX40L is highly expressed in the atherosclerotic plaque. The ligation of OX40 and OX40L can induce increased expression of c-jun gene and RANTES/CCL5 in human umbilicus veins endothelial cells , directly induces the adhesion of T-cells or OX40L+T cells to endothelial cells. Previous study find that the C57BL/6 mice are atherosclerosis susceptible, while the C3H/ He mice are atherosclerosis resistant. They foud the first atherosclerosis susceptible gene including OX40L gene by the mothed of quantitative trait locus. The dysfunction of endothelial cells not only is the initial step of development of atherosclerosis but also accelerate the formation of the atherosclerosis lesions. While the atherosclerosis lesions are relieve from the protection of endothelial cells The administration of OX40L monoclonal antibody reverses the plaque in atherosclerotic mice. These data suggests a pivotal role of OX40L in atherosclerosis. C-reactive protein (CRP) is an acute-phase reactant and serves as a pattern-recognition molecule in the innate immune system. It is regarded as an inflammatory marker atherosclerosis. Our previous data showed that CRP can induce the injury of cardiac myocytes. In fact, the high expression of CRP level is seen in the blood of patients with coronary artery disease. Whether the increase of CRP in the peripheral circulation influences the expression of OX40L in endothelial cells is unknown.ObjectiveThe present study was designed to compare the expression of OX40L in tissues from normal atherosclerosis susceptible C57BL/6 mice with normal atherosclerosis resistant BALB/c mice. Investigate the effect of CRP on OX40L expression in cultured C57BL/6 mice aorta endothelial cells.Methods1.Eight C57BL/6 mice and eight BALB/c mice at the age of eight weeks were sacrificed, and the total mRNA as well as tissue homogenates were isolated from the heart, brain, kidney, skeletal muscle and spleen. The OX40L mRNA expression level detected by RT-PCR and the protein expression level was detected by Western Blot.2.CRP was purified by Immobilized p-Aminophenyl Phosphoryl Choline Gel. The purified CRP was analysis by SDS-PAGE and identified by Western Blot.3. BALB/c mice were sacrificed and the aorta was dissected. The generation of cultured endothelial cells was utilized by collagenaseⅡdigestion of the aorta. The identification of the endothelial cells carried out by immunostaining with special antibody factorⅧantibody. When the endothelial cells grow to about 60~70% of the dish, 100ug/ml CRP was applied to medium. 48 hours later, the endothelial cells were collected and the total RNA and protein homogenates were isolated. The gene expression and protein expression of OX40L were measured by RT-PCR and Western Blot respectively.Results1. The OX40L mRNA expression level in the heart of C57BL/6 mice was significantly higher than that of BALB/c mice (0.79±0.10 vs 0.60±0.05, P <0.05, n=8). There was no significant difference between BALB/c mice and C57BL/6 mice in brain, kidney and skeletal muscle. The OX40L mRNA level was augmented significantly in the spleen from BALB/c mice than that from C57BL/6 mice (0.39±0.17 vs 0.25±0.04, P <0.05, n=8). The protein levels of OX40L expressed significantly higher in the heart, brain and kidney from and was C57BL/6 mice than those from BALB/c mice, respectively (0.56±0.01 vs 0.19±0.07, 0.18±0.07 vs 0.01±0.01, 0.31±0.13 vs 0.03±0.01, P<0.05, n=8). No significant difference was obtained in skeletal muscle and spleen between these two kinds of mouse.2. The purified CRP showed more than 95% purity by SDS-PAGE. The purified CRP binded specifically to CRP monoclonal antibody.3. After 48 hours treatment of 100 ug/ml CRP, the expressions of OX40L mRNA or protein level, in the treated cultured endothelial cells were significantly augmented than those in the untreated cultured endothelial cells (0.09±0.04 vs 0.76±0.12 and 0.07±0.02 vs 0.21±0.06,P<0.05, n=3).ConclusionsThe present study showed the different expression level of OX40L tissues from BALB/c mice and C57BL/6 mice. CRP can induce the expression of OX40L in mouse aortic endothelial cells. These data suggested a role underlying the mechanism of OX40L in atherosclerosis.更多还原