【作者】 刘强；

【导师】 杨笛；

【作者基本信息】 南京医科大学 ， 内科学， 2011， 硕士

【摘要】 背景近年来OX40/OX40L受体-配体信号途径参与动脉粥样硬化的发生、发展过程逐渐受到关注。共刺激分子OX40配体（OX40L/CD134L/gp34）,是参与免疫反应的辅助性分子,属肿瘤坏死因子（TNF）家族成员。OX40L主要表达在活化的抗原呈递细胞表面,如树突状细胞、B淋巴细胞、巨噬细胞,血管内皮细胞以及心脏、骨骼肌和肺等组织中。OX40L的受体OX40主要表达在活化的T细胞表面。OX40L与OX40结合后,通过OX40信号通路,促进活化后T细胞的存活、增殖和记忆性T细胞的产生,增强T细胞与其它细胞的粘附,传递抗原刺激信号,参与细胞的免疫活化过程。阻断OX40/OX40L作用不抑制全身免疫反应,成为治疗慢性炎症性疾病的目标之一。2005年,美国Jackson实验室首次报告了OX40L参与动脉粥样硬化的发生,抗OX40L抗体干预可抑制斑块形成、促进斑块稳定。在主动脉动脉粥样硬化斑块部位的内皮细胞、平滑肌细胞、巨噬细胞和淋巴细胞均有丰富的OX40L表达。斑块部位OX40L的局灶性表达增高,是仅仅增强共刺激分子信号的作用,还是另有其它的生物学效应?在斑块破裂中是否发挥作用?尚待研究。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。内皮细胞OX40L的一个重要特性是可直接介导T细胞或OX40+转染的T细胞向内皮细胞黏附,斑块内有大量T细胞集聚,斑块部位内皮细胞OX40L表达增高和OX40L逆向信号的激活,增强了内皮细胞与T细胞间的信息传递,导致内皮细胞内促斑块破裂因子与斑块稳定因子表达与释放的失平衡,可能是斑块破裂的触发因素之一。细胞内游离钙浓度([Ca²⁺]_i)在内皮细胞的信号转导中起重要作用,内皮细胞[Ca²⁺]_i水平升高可以促进多种促栓塞和缩血管物质从内皮细胞释放,如内皮素、vWF、组织因子、前列腺素E2等。生理条件下,内皮细胞以动态方式维持[Ca²⁺]_i的平衡,通过抑制这些血管活性物质从内皮细胞分泌而维持内皮细胞的抗栓、非粘附等特性。TNF配体胞浆区氨基酸序列高度保守,其逆向信号的激活可诱导细胞内钙离子浓度增高。OX40L的逆向信号是否影响内皮细胞[Ca^{2 +}]_i的改变,尚不清楚。本论文将研究OX40L逆向信号的激活对动脉内皮细胞[Ca^{2 +}]_i的作用,为OX40/OX40L信号致血管病变机制提供实验数据。目的研究OX40L逆向信号对C57BL/6J小鼠主动脉内皮细胞胞内游离钙浓度的作用。方法1、内皮细胞培养8～12周的C57BL/6J小鼠,腹腔注射肝素钠1250U/只,20分钟后用7.5%的水合氯醛腹腔注射麻醉（约11.25g/kg体重）。依次切开皮肤、肌层、筋膜,暴露心肺和胸、腹主动脉,肾动脉分叉处剪断腹主动脉放血后,用1ml无菌注射器自左室心尖部灌注含1000U/ml肝素钠的磷酸盐缓冲液,冲洗主动脉至流出液变清亮为止。用血管夹钳夹血管一端,自另一端注入Ⅱ型胶原酶消化液,37℃消化45 min。用5 ml 20%胎牛血清-DMEM冲洗血管终止消化,收集冲洗液,1000 rpm/min离心5 min。调整细胞密度至10⁵/ml后接种至I型鼠尾胶原包被的六孔板,添加1～2ml的内皮细胞培养液,5%CO₂孵育箱内37℃贴壁2 h。吸去未贴壁细胞及内皮细胞培养液,加1ml内皮细胞培养液,5%CO2,37℃孵育箱内培养3天。每2～3天更换培养液一次。2、内皮细胞鉴定当细胞呈汇合状态时取出备用,用冷甲醇固定细胞抗原,洗脱液细胞膜打孔,0.6% H₂O₂孵育灭活内源性过氧化物酶,封闭液封闭非特异性抗原。加入vWF抗体（1:400）、α- actin抗体（1:150）,4℃过夜。加入辣根过氧化酶标记的anti-rabbit二抗（1:200）,37℃反应60min。加入DAB反应液显色,观察阳性染色细胞的比例。3、内皮细胞OX40L表达细胞呈汇合状态时取出,100%冷甲醇﹣20℃固定细胞抗原20 min。磷酸盐缓冲液清洗后,室温下洗脱液细胞膜打孔15 min,随后用0.6% H₂O₂室温孵育15 min。磷酸盐缓冲液清洗后,用封闭液室温孵育45 min。加入OX40L抗体（1:200）,4℃过夜。磷酸盐缓冲液清洗后,加入辣根过氧化酶标记的anti-goat二抗（1:200）,37℃,60min。加入DAB反应液后避光反应2min左右,用磷酸盐缓冲液终止反应,观察阳性染色细胞的比例。4、OX40L逆向信号激活对内皮细胞[Ca²⁺]_i的作用将接种在激光共聚焦培养皿上的同批次内皮细胞,用DMEM培养24h,细胞分组（1）观察OX40对内皮细胞[Ca²⁺]_i的作用:内皮细胞分为对照组（内皮细胞未用OX40干预）、OX40干预组（内皮细胞分别用25 ug/L,50 ug/L,75 ug/L,100 ug/L和125 ug/L OX40于37℃孵育30min）;（2）观察OX40动态干预对内皮细胞[Ca²⁺]_i的作用:内皮细胞分为对照组（内皮细胞未用OX40干预）、OX40不同时间干预组（将100ug/L OX40与内皮细胞分别孵育24h、48h和72h）;（3）观察OX40L信号阻断的作用:内皮细胞用100 ug/L羊抗鼠OX40L单克隆抗体37℃预孵育30min,100 ug/L OX40,在37℃孵育30min。各组细胞吸去培养液,用DMEM清洗2次,加入钙离子荧光探针Fura-2/AM（终浓度为4μM）,于37℃5%CO₂负载30 min,吸去含Fura-2/AM的上清液,用37℃清洗液清洗3次。[Ca²⁺]_i测定前将细胞在37℃复温5min,置于倒置显微镜下,选择细胞间分界清楚及细胞形态良好的内皮细胞,调整曝光时间。以340 nm和380 nm为波长激发,510 nm为波长发射,激发波长变换时间设定为124ms,采集图像,扫描24次,记录24个荧光强度数值。采用TILL VISIDN软件系统进行数据处理和分析,计算荧光比值（R= F340/F380）计算[Ca²⁺]_i的相对浓度。结果1.内皮细胞从主动脉条中分离出来时呈圆形小滴,3天左右可见细胞集落形成,集落中央为圆形细胞群,周边有梭形细胞不断生成。在培养的7～10天,内皮细胞分界清楚,以类圆形和多角形细胞为主,也有梭形细胞,胞浆丰富,胞核呈圆形或椭圆形,偶见双核。并逐渐汇合成单层,细胞大小均匀,排列紧密,互不重叠,呈现内皮细胞典型的“鹅卵石”样排列特征。内皮细胞表面标志物vWF免疫细胞化学染色呈棕黄色阳性反应,平滑肌细胞标志物alpha -actin染色和阴性对照组染色均不染色。在倒置显微镜下随机选取5个视野计数细胞,所见细胞为vWF阳性染色细胞。2.培养8～10天的小鼠主动脉内皮细胞,行OX40L免疫细胞化学染色,?胞浆被染成棕黄色,胞核无染色,呈阳性反应。在倒置显微镜下随机抽取5个视野计数细胞,所见细胞为OX40L阳性表达细胞,即在培养的小鼠主动脉内皮细胞检测到OX40L的表达。3. OX40L逆向信号对C57BL/6J小鼠主动脉内皮细胞胞内游离钙浓度的影响:?3.1 25ug/L以下的OX40刺激内皮细胞后不引起[Ca²⁺]_i改变（P>0.05）;OX40在50～125 ug/L浓度范围内,可使C57BL/6J小鼠主动脉内皮细胞[Ca²⁺]_i水平升高并呈剂量依赖关系。在100ug/L时[Ca²⁺]_i达到峰值,125ug/L较100ug/L OX40胞内钙有缓慢下降的趋势但未达统计学差异（P>0.05）。3.2单独加入OX40阻滞剂anti-OX40L抗体前后, [Ca²⁺]_i无明显差别（P>0.05）。先用100ug/L的anti-OX40L抗体预处理30min再加入OX40 （25ug/L）,与单独加OX40（25ug/L）相比, [Ca²⁺]_i无明显差别（P>0.05）。先用anti-OX40L抗体（100ug/L）预处理30min再加入OX40 （50,75,100ug/L）,与单独加OX40（50,75,100ug/L）相比, [Ca²⁺]_i浓度明显降低（P<0.05）。3.3 100ug/L的OX40刺激内皮细胞24h,与对照组相比,内皮细胞[Ca^{2 +}]_i无明显改变（P>0.05）;100ug/L的OX40刺激内皮细胞48h、72h,与对照组相比,内皮细胞[Ca²⁺]_i明显增高（P<0.05）。结论1.应用建立的小鼠主动脉内皮细胞体外培养方法,观察到培养的内皮细胞汇合成单层时,细胞大小均匀,排列紧密,互不重叠,呈现内皮细胞典型的“鹅卵石”样排列特征。阴性对照无显色反应和平滑肌细胞表面标志物α- actin染色无显色反应,表明培养的细胞为内皮细胞。2.用免疫细胞化学染色法证实在培养的小鼠主动脉内皮细胞上有OX40L表达。3. OX40激活OX40L逆向信号使内皮细胞[Ca²⁺]_i升高,呈浓度和时间相关性;用抗OX40L单克隆抗体阻断OX40与OX40L结合,抑制了OX40引起的内皮细胞[Ca²⁺]_i升高,提示OX40L逆向信号的激活引起内皮细胞[Ca²⁺]_i升高是经OX40L跨膜蛋白分子介导的。更多还原

【Abstract】 BackgroundCo-stimulatory molecules OX40/OX40L play an important role in mediation signal transduction in the development and process of atherosclerosis. OX40 ligand （OX40L/CD134L/gp34）, an accessory molecule of immune response, belongs to the TNF super-family. OX40L is primarily found on activated antigen presenting cell, including dendritic cells、B-lymphocyte、macrophages、vascular endothelial cells、heart、skeletal muscle and lung. The receptor of OX40L, OX40, is primarily induced on activated T cells. OX40/OX40L mediated co-stimulation signal maintains T cells survival; promotes T cells proliferation and generation of memory T cells; enhances adhesion between T-cells and other cells; transmits stimulant information of antigens; participates process of cells immune activation. Interruption of OX40/OX40L interaction has been shown to ameliorate inflammatory reaction in the inflamed site instead of global inflammatory reaction. Hence, it’s an excellent target for inflammatory-mediated diseases therapy.Up to 2005, Jackson Laboratory firstly reported that OX40/OX40L participate in atherosclerotic disease process. Anti-OX40L antibody treatment leaded to a marked decrease of atherosclerotic plaque and promoted plaque stabilization. The expression of OX40L was found in all the major cell types in atherosclerotic lesions, including endothelial cells, smooth muscle cell, macrophages, T-lymphocytes. OX40L is expressed more highly in plaque tissue than in normal. Apart from the role to deliver costimulatory signals, are there other biological effects exist? What’s the role in atherosclerotic plaque rupture? OX40/OX40L, a pair of co-stimulatory molecules, generates bi-directional signals and may elicit respective biological effects. It is an important characteristic that ECs OX40L directly mediates T-cells or OX40+ T cells adhesion to ECs and T-lymphocytes gather at atherosclerosis plaques. Enhanced expression of ECs OX40L in plaque and activation of OX40L reverse signal potentiates signal transduction between T cells and ECs, which could lead to the disequilibrium of expression and secretion between plaque stable factor and plaque unstable factor.Elevated cytosolic free Ca²⁺ is a trigger in many signal transduction, which could lead to the secretion of a variety of endothelium-derived atherogenic factors and vasoconstrictor. For instance, endothelin、von willebrand factor、tissue factor and prostaglandin E2. These factors have been confirmed harmful, causing inflammation, increasing plaque instability. OX40, a known proinflammatory factors, maybe involved in [Ca²⁺]_i overproduction. Hence, we investigated the changes of [Ca²⁺]_i in MAECs subsequently.ObjectThe present study was designed to investigate the effect of intracellular free calcium concentration of aortic endothelial Cells of C57BL/6J mice after activation of OX40L reverse signal pathway.Materials and methods1. C57BL/6J Mice, aged 8 to 12 weeks, was treated with an intraperitoneal injection of heparin sodium （1250U）, and then anesthetized with 11.25g/kg chloral hydrate. The midline of the abdomen is incised. Then the thorax was opened to expose the thoracic aorta and abdominal aorta. The abdiminal aorta is cut at the site of renal artery to release the blood, and then perfused with 1 ml phosphate buffered saline containing 1000U/ml of heparin from the apex of left ventricle. The thoracic aorta was cannulated by a 23-gaug cannula. Then the thoracic aorta was filled with collagenase type II solution to dissociate for 45 min at 37°C. Collagenase action was stopped and detached cells were obtained by perfusing the thoracic aorta with DMEM supplemented with 20% fetal calf serum and 1% penicillin-streptomycin. The perfusate was centrifuged at 1000rpm for 5 min and the pellet was resuspended with endothelial cell culture media and maintained in the same solution and cultured in 6 well-plate coated with collagen type I in a humidified atmosphere of 5% CO₂ at 37°C. The medium was removed after 2 h and covered with fresh culture medium for 3 days. Fresh endothelial cells culture medium was changed every 2 to 3 days.2. Confluent monolayers were fixed with chilled methanol, and endogenous peroxidases were quenched by 0.06% H₂O₂. Cells were subsequently blocked with goat serum used to generate the secondary antibody. ECs were characterization using anti-vWF antibody （1:400 dilution） and smooth muscle cells were detected by an antibody against alpha actin（1:150 dilution）. Primary antisera were incubated at 4°C overnight, followed by an incubation of a secondary horseradish peroxidase （HRP） conjugated antibody （1:200dilution） under 37°C for 1h. Immunoreactivity was visualized by incubation with the DAB kit.3. Confluent monolayers were fixed with chilled methanol for 20min at -20°C, Endogenous peroxidases were blocked by 15min incubation in 0.06% H₂O₂. Samples were subsequently blocked with serum used to generate the secondary antibody. We then applied an antibody against mouse OX40L （1:200dilution）. The specific binding was detected with HRP labeled secondary antibody （1:200 dilution） and DAB as a substrate. The cells staining images were produced in an OLYMPUS inverted microscope equipped with a Cannon digital camera and underwent adjustment of brightness and contrast using Adobe Photoshop 7.0.4. ECs were assigned to these groups: 1) ECs, not receiving any treatment, was performed as control. 2) ECs, subjected to indicated OX40 concentrations stimulation （0, 25, 50, 75, 100, 125 ug/L） at 37°C for 30min, were used to investigate the dose-dependent manner of OX40-induced [Ca²⁺]_i ;3) ECs, pretreated with the selective and competive OX40 inhibitor anti-OX40L antibody （100ug/L） prior to adding OX40（0, 25, 50, 75, 100ug/L）, were used to investigate the down-regulation of OX40 stimulation. 4) ECs, subjected to indicated stimulation time （24h, 48h,72h） at 37°C, were performed to investigate the time manner of OX40-induced[Ca²⁺]_i. Then the cells were loaded with 4umol/L calcium sensitive probe Fura-2/AM for 30 min at 37°C in the dark. Under inverted microscope: the excitation wavelengths were 340 and 380 nm, and the emitted fluorescence was measured at 510 nm. Intracellular free calcium was analyzed as the ratio of background-corrected fluorescence at 340 nm excitation to 380 nm excitation （F340/F380 ratio）. Changes in the ratio of F340/F380 were regarded as changes in [Ca²⁺]_i.Results1. Fresh cells effluents contained small clumps is rounded droplet, which spread to form small epithelioid clusters within the first 3d in culture. These clusters increased in size and gradually coalesced to form incomplete mono-layers by 7-10 days. Cells within these early mono-layers were uniform in appearance: elongated, with single ovoid nuclei containing one to two prominent nucleoli, and a broad, thin peripheral cytoplasm with distinct borders. Then, single cell-thick confluent mono-layers of densely packed polygonal cells had formed, presenting with a cobblestone appearance, a characteristic morphology of ECs. Immunocytochemical characterization of cells showed a positive staining for vWF whereas alpha-actin staining and negative control staining was negative.2. OX40L was expressed on cultured vascular endothelial cells by ECs OX40L immunocytochemical staining. Under inverted microscope, all ECs were OX40L positive staining cells in 5 random visual fields.3. Effects of OX40L reverse signal pathway on intracellular calcium concentration in aortic endothelial Cells of C57BL/6J mice:3.1 When cells were subjected to 25ug/L OX40 stimulation at 37°C for 30min, there was no change in [Ca²⁺]_i of ECs （P>0.05）. The OX40 increased [Ca²⁺]_i was observed at 50ug/L and significantly increased in dose-dependent manner from 50ug/L to 125ug/L （（P<0.05））. And there was a peak at 100ug/L OX40 stimulation. However, there was no difference in [Ca²⁺]_i of ECs between 100ug/L and 125ug/L（P>0.05）.3.2 There was no difference in[Ca²⁺]_i before or after adding anti-OX40L antibody（P>0.05）. However, in the selective OX40 inhibitor anti-OX40L antibody pretreated cells （100ugl/L for 30min） prior to adding OX40 （50,75 , 100ug/L）, this increase of[Ca²⁺]_i was significantly abrogated（P<0.05）.3.3 When cells were subjected to 100ug/L OX40 stimulation at 37°C for different times （24h, 48h, 72h）, there was no change in [Ca²⁺]_i of ECs at 24h（P>0.05）. [Ca²⁺]_i of EC significantly increased after the treatment of OX40 in 48h and 72h（P<0.05）.Conclusions:1. The present study established a method to isolate and culture aortic endothelial cells from mice which was confirmed by endothelial cell morphology and immunocytochemical staining.2. The cultured aortic endothelial cells from C57BL/6J mice expressed OX40L.3. The results indicated that the activation of OX40L reverse signal can increase the intracellular free calcium concentration of mouse aortic endothelial cells. The elevated of intracellular free calcium can partly explain the effect of OX/OX40L on the pathyology of vascular disorder.更多还原