关键词： 猪血凝性脑脊髓炎病毒   ATF3   MX1   小鼠  

摘要： 猪血凝性脑脊髓炎是由猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus, PHEV)引起仔猪的一种急性、高度接触性传染病,主要侵害1～3周龄哺乳期的仔猪,临床以仔猪呕吐、衰竭和/或明显的神经症状为主要特征,死亡率达20%～100%,给养殖业带来巨大的经济损失。PHEV具有嗜神经性,主要由感染部位经外周神经向中枢神经系统传播,引起大脑呈现非化脓性脑脊髓炎的病理性损伤。MX1蛋白是一种抗病毒蛋白,由Ⅰ型干扰素(IFNα/β)诱导宿主细胞产生的,具有广谱的抗病毒作用。ATF3是激活转录因子家族成员,是转录调控、细胞凋亡、细胞分裂和生存的一个重要调控因子,可以使细胞增殖加速,与病毒的复制密切相关。本实验室前期研究表明,小鼠大脑皮层及神经瘤细胞N2a感染PHEV后,ATF3基因与MX1基因均呈差异表达。但这两种基因差异表达是否与病毒感染有直接的关系,其表达水平与PHEV病毒载量之间是否有相关性尚不明确。因此,本研究旨在通过检测PHEV感染后大脑中ATF3和MX1的表达水平,探索ATF3、MX1表达水平和PHEV病毒载量之间的关系,为阐明抗病毒蛋白和活性转录因子的作用机理奠定一定的理论基础。方法：建立PHEV急性感染小鼠模型,用0.1mL的PHEV(103TCID50/0.1mL)以颅内接种方式感染5周龄的雌性健康昆明小鼠,使小鼠在第5d全部死亡。之后将30只小鼠随机分为病毒组和对照组,每组各15只,病毒组颅内注射0.1mL的PHEV,对照组接种相同剂量的PBS。在感染后第1～5d每天每组分别随机选3只小鼠剖杀,取大脑一部分于液氮中保存,进行荧光定量PCR;另一部分大脑固定于福尔马林溶液中,进行H.E.染色和免疫组织化学试验。结果发现,接毒后第1～2d的小鼠精神状态及饮食无明显异常;接毒后第3d小鼠出现明显的神经症状,如弓背,两前肢呈“弹钢琴样”上下摆动;接毒后第5d小鼠瘫痪,抽搐死亡;对照组小鼠无异常反应。剖检发现接毒组小鼠大脑充血水肿;组织病理学观察发现接毒组小鼠大脑呈典型的非化脓性脑炎变化,如神经元固缩、坏死,小胶质细胞增多,卫星现象及“血管套”形成。Real-time PCR结果显示,接毒组小鼠大脑ATF3和MX1基因表达水平与PHEV病毒载量均升高,且ATF3和MX1基因表达水平与PHEV的病毒载量呈现正相关关系,相关性极显著(p<0.01)。对不同感染时间段小鼠大脑PHEV病毒载量和ATF3基因表达水平进行比较,结果显示,对照组、感染第1～4d小鼠与第5d小鼠之间病毒载量和ATF3基因表达水平均呈现显著差异(p<0.05)。免疫组化结果显示,小鼠感染病毒后大脑ATF3、MX1蛋白及PHEV的胞浆中均有棕黄色阳性信号,且大脑中ATF3蛋白、MX1蛋白表达量和PHEV均增加。

关键词： 重组高密度脂蛋白   炎症反应   脂多糖   RAW264.7   细胞   ATF3  

摘要： 背景:ATF3在抑制炎症信号中通过抑制TLR的产生而发挥重要作用,高密度脂蛋白(HDL)或其主要蛋白成份Apo A-I具有显著的抗炎症作用。本研究探讨载脂蛋白A-I(Apo A-I)及其半胱氨酸突变体重组高密度脂蛋白对ATF3表达量的影响。方法:(1)利用pET原核表达系统表达载脂蛋白A-I74,A-I228,A-Im和A-IWT,亲和层析纯化后,加入DPPC,制作成重组高密度脂蛋白。(2)培养RAW264.7细胞,LPS刺激诱导炎症细胞模型,然后给于重组高密度脂蛋白干预,ELISA试剂盒检测炎症因子IL-6和TNF-α的表达水平,观察细胞形态学的改变,RT-PCR和western blotting检测细胞中转录激活因子ATF3 m RNA和蛋白表达水平的变化。结果:细胞形态学观察发现,与正常组比较,LPS组与rHDL228组细胞形态趋向于巨噬细胞化并出现降解,rHDL74、rHDLM和rHDLWT对LPS刺激的细胞有不同的保护作用。ELISA检测发现,rHDL74、rHDLm和rHDLwt中TNF-ɑ与IL-6的表达水平恢复到接近空白对照组水平,rHDL 228组TNF-ɑ与IL-6水平较较空白对照组明显升高(p<0.01)。rHDL74、rHDLM和rHDLWT中TNF-ɑ与IL-6水平较LPS组明显降低(P<0.01)。Western blotting结果显示,与对照组相比,LPS组与rHDL 228组ATF3的表达水平下降,具有显著性差异(p<0.01)。rHDL74、rHDLM和rHDLWT组表达水平明显升高且差异显著(p<0.05)。结论:重组高密度脂蛋白rHDL74、rHDLWT、rHDLM促进ATF3在m RNA和蛋白水平上的表达。而rHDL228下调其表达。

关键词： 急性肺损伤   脂多糖   活化转录因子3   TL1A   RNA干扰   基因芯片  

摘要： 1.背景和目的急性肺损伤(acute lung injury,ALI),急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是因为各种肺内、外因素所致的以肺泡毛细血管膜弥漫性损伤、严重低氧血症为特征的呼吸危重症,因外其发病机制尚不完全清楚,所以仍有较高的病死率。其中,炎症反应失衡在疾病的发生发展中占有重要地位。由革兰阴性杆菌感染引起的脓毒血症是导致ALI/ARDS的重要病因,而脂多糖(Lipopolysaccharide LPS)作为革兰阴性杆菌的胞壁成分是先天性免疫反应及炎症反应的启动子,是导致ALI/ARDS的重要原因之一。因此,明确LPS导致炎症损伤的发病机制对急性肺损伤的治疗有重要意义。目前已证实,转录活化因子3(activating transcription factor 3,ATF3)是一应激早期快反应基因,其表达与炎症反应、机体稳态维持、肿瘤发生等密切相关。在多种炎症模型中,ATF3对炎症反应起刹车作用。然而,ATF3在LPS致急性肺损伤中的作用尚不完全清楚。鉴于此,本课题拟通过比较腹腔注射LPS后的野生型小鼠和ATF3敲基因小鼠的各项炎症指标,明确ATF3在LPS致急性肺损伤小鼠模型中的作用;将两组小鼠肺组织通过基因芯片技术寻找其差异基因;并最后通过构建干扰质粒下调ATF3在小鼠单核巨噬细胞系RAW264.7细胞中的表达,探讨ATF3调控LPS导致炎症反应的可能机制,从而为急性肺损伤的防治提供潜在的新靶点。2.研究方法(1)构建急性肺损伤动物模型,并明确ATF3在动物模型中的表达规律向小鼠进行腹腔注射LPS,注射量为15mg/kg,从而建立急性肺损伤小鼠的动物模型,分别采取RT-PCR以及Western blot的方法,检测ATF3在LPS刺激后的各个时相点,小鼠肺组织中的表达情况,从而明确其表达规律。(2)观察ATF3对LPS诱导的急性肺损伤小鼠的保护作用构建ATF3敲基因小鼠,用Western blot的方法鉴定敲基因小鼠中ATF3的表达缺失,通过比较野生型小鼠和ATF3敲基因小鼠的肺组织病理切片、肺组织湿干比、肺组织中炎症因子的表达水平、肺泡灌洗液蛋白浓度以及7天存活率来评估ATF3对LPS诱导的急性肺损伤小鼠的保护作用。(3)利用基因芯片技术筛选急性肺损伤小鼠中与atf3相关的差异基因同时用lps对c57小鼠及atf3敲基因小鼠进行腹腔注射后2小时,然后提取两组小鼠肺组织的总rna,通过基因芯片技术筛选差异表达基因。再结合文献阅读,最终重点关注tl1a分子,并提出atf3可能是通过下调tl1a的表达实现对急性肺损伤的炎症保护作用这一假说。(4)以小鼠单核巨噬细胞株raw264.7细胞为模型探讨atf3在lps诱导的急性肺损伤中的可能机制用rt-pcr和westernblot的方法比较raw264.7细胞中,atf3在lps刺激后各个时间点的表达变化。构建质粒,用rna干扰的方法下调atf3在raw264.7细胞中的表达,观察atf3对lps诱导的细胞炎症损伤以及信号分子通路tl1a表达的影响;采用sirna双干扰技术同时下调atf3和tl1a在raw264.7细胞中的表达后,通过检测炎症因子il-6的表达变化说明tl1a信号通过在atf3调控lps诱导的急性肺损伤中的作用。3.结果(1)atf3表达于正常小鼠肺组织,lps腹腔注射后,小鼠肺组织内atf3的表达水平明显增加(p<0.05),以lps腹腔注射2h时相点达表达高峰,rt-pcr与westernblot的结果趋势一致。(2)将野生型小鼠与atf3敲基因小鼠作为对比,lps腹腔注射以后,atf3敲基因小鼠的肺组织病理损伤加重,肺组织湿干比增加,炎症因子il-6、il-1β、tnf-α表达增加,肺泡灌洗液蛋白浓度增加,小鼠7天存活率显著下降(p<0.05)。(3)从基因芯片的结果中找到了atf3敲基因小鼠和野生型小鼠的差异基因tl1a,结合阅读文献,提出了atf3可能通过下调tl1a表达,对lps诱导的急性肺损伤炎症起到调控作用的科学假说。(4)lps刺激后,atf3在raw264.7细胞中的表达上调,表达高峰点在lps处理6h后,下调atf3的表达会增加lps诱导的炎症因子tnf-α和il-6的释放;与lps和ncsirna+lps组相比,atf3sirna组和atf3sirna+lps组raw264.7细胞中tl1a的表达显著增加(p<0.05),为进一步证实tl1a信号通路在atf3调控lps诱导的急性肺损伤中的作用,我们同时下调了atf3和tl1a在raw264.7细胞中的表达,与atf3sirna+lps组相比,atf3sirna+tl1asirna组raw264.7细胞中il-6的表达

关键词： GROWTH-FACTOR-BETA   NECROSIS-FACTOR-ALPHA   TRIGLYCERIDE-RICH LIPOPROTEINS   LOW-DENSITY-LIPOPROTEIN   TRANSCRIPTIONAL REPRESSOR ATF3/LRF1   CEREBELLAR GRANULE NEURONS   EPITHELIAL-CELLS   GENE-EXPRESSION   TRANSFORMING GROWTH-FACTOR-BETA-1   C-JUN  

摘要： Studies have suggested a link between the transforming growth factor beta 1 (TGF-beta 1) signaling cascade and the stress-inducible activating transcription factor 3 (ATF3). We have demonstrated that triglyceride-rich lipoproteins (TGRL) lipolysis products activate MAP kinase stress associated JNK/c-Jun pathways resulting in up-regulation of ATF3, pro-inflammatory genes and induction of apoptosis in human aortic endothelial cells. Here we demonstrate increased release of active TGF-beta at 15 min, phosphorylation of Smad2 and translocation of co-Smad4 from cytosol to nucleus after a 1.5 h treatment with lipolysis products. Activation and translocation of Smad2 and 4 was blocked by addition of SB431542 (10 mu M), a specific inhibitor of TGF-beta-activin receptor ALKs 4, 5, 7. Both ALK receptor inhibition and anti TGF-beta 1 antibody prevented lipolysis product induced up-regulation of ATF3 mRNA and protein. ALK inhibition prevented lipolysis product-induced nuclear accumulation of ATF3. ALKs 4, 5, 7 inhibition also prevented phosphorylation of c-Jun and TGRL lipolysis product-induced p53 and caspase-3 protein expression. These findings demonstrate that TGRL lipolysis products cause release of active TGF-beta and lipolysis product-induced apoptosis is dependent on TGF-beta signaling. Furthermore, signaling through the stress associated JNK/c-Jun pathway is dependent on TGF-beta signaling suggesting that TGF-beta signaling is necessary for nuclear accumulation of the ATF3/cJun transcription complex and induction of pro-inflammatory responses.

关键词： CARDIOMYOCYTES  

关键词： VENTRICULAR remodeling   MACROPHAGES   MANUSCRIPTS   PHENYLEPHRINE (Drug)   CARDIAC hypertrophy   MICE as laboratory animals  

关键词： ATF3  

关键词： Network analysis   Gene expression analysis   Iterative method   Partial least square   Gene regulation   Dynamic modeling   TRANSCRIPTION FACTOR ACTIVITIES   BREAST EPITHELIAL-CELLS   ADAPTIVE-RESPONSE GENE   MICROARRAY DATA   REGULATORY NETWORKS   EXPRESSION DATA   CANCER   ATF3   ACTIVATION   ALGORITHM  

摘要： Background: Network component analysis (NCA) became a popular tool to understand complex regulatory networks. The method uses high-throughput gene expression data and a priori topology to reconstruct transcription factor activity profiles. Current NCA algorithms are constrained by several conditions posed on the network topology, to guarantee unique reconstruction (termed compliancy). However, the restrictions these conditions pose are not necessarily true from biological perspective and they force network size reduction, pruning potentially important components. Results: To address this, we developed a novel, Iterative Sub-Network Component Analysis (ISNCA) for reconstructing networks at any size. By dividing the initial network into smaller, compliant subnetworks, the algorithm first predicts the reconstruction of each subntework using standard NCA algorithms. It then subtracts from the reconstruction the contribution of the shared components from the other subnetwork. We tested the ISNCA on real, large datasets using various NCA algorithms. The size of the networks we tested and the accuracy of the reconstruction increased significantly. Importantly, FOXA1, ATF2, ATF3 and many other known key regulators in breast cancer could not be incorporated by any NCA algorithm because of the necessary conditions. However, their temporal activities could be reconstructed by our algorithm, and therefore their involvement in breast cancer could be analyzed. Conclusions: Our framework enables reconstruction of large gene expression data networks, without reducing their size or pruning potentially important components, and at the same time rendering the results more biological plausible. Our ISNCA method is not only suitable for prediction of key regulators in cancer studies, but it can be applied to any high-throughput gene expression data.

关键词： ACTIVATING TRANSCRIPTION FACTOR-3   I INTERFERON RECEPTOR   ANTIVIRAL RESPONSE   GENE-EXPRESSION   NEGATIVE REGULATION   SIGNALING AXIS   FACTOR-BINDING   BETA   25-HYDROXYCHOLESTEROL   ALPHA  

摘要： Cytokines and IFNs downstream of innate immune pathways are critical for mounting an appropriate immune response to microbial infection. However, the expression of these inflammatory mediators is tightly regulated, as uncontrolled production can result in tissue damage and lead to chronic inflammatory conditions and autoimmune diseases. Activating transcription factor 3 (ATF3) is an important transcriptional modulator that limits the inflammatory response by controlling the expression of a number of cytokines and chemokines. However, its role in modulating IFN responses remains poorly defined. In this study, we demonstrate that ATF3 expression in macrophages is necessary for governing basal IFN-beta expression, as well as the magnitude of IFN-beta cytokine production following activation of innate immune receptors. We found that ATF3 acted as a transcriptional repressor and regulated IFN-beta via direct binding to a previously unidentified specific regulatory site distal to the Ifnb1 promoter. Additionally, we observed that ATF3 itself is a type I IFN-inducible gene, and that ATF3 further modulates the expression of a subset of inflammatory genes downstream of IFN signaling, suggesting it constitutes a key component of an IFN negative feedback loop. Consistent with this, macrophages deficient in Atf3 showed enhanced viral clearance in lymphocytic choriomeningitis virus and vesicular stomatitis virus infection models. Our study therefore demonstrates an important role for ATF3 in modulating IFN responses in macrophages by controlling basal and inducible levels of IFN beta, as well as the expression of genes downstream of IFN signaling.

关键词： Pressure overload   Cardiac remodeling   Macrophages   Bone marrow transplantation   Cardiac hypertrophy   ACTIVATING TRANSCRIPTION FACTOR-3   JUN DIMERIZATION PROTEIN-2   MYOCARDIAL FIBROSIS   HYPERTROPHIC GROWTH   ADAPTIVE-RESPONSE   C-JUN   ATF3   EXPRESSION   REPRESSOR   AP-1  

摘要： Rationale: Pressure overload induces adaptive remodeling processes in the heart. However, when pressure overload persists, adaptive changes turn into maladaptive alterations leading to cardiac hypertrophy and heart failure. ATF3 is a stress inducible transcription factor that is transiently expressed following neuroendocrine stimulation. However, its role in chronic pressure overload dependent cardiac hypertrophy is currently unknown. Objective: The objective of the study was to study the role of ATF3 in chronic pressure overload dependent cardiac remodeling processes. Methods and results: Pressure overload was induced by phenylephrine (PE) mini-osmotic pumps in various mice models of whole body, cardiac specific, bone marrow (BM) specific and macrophage specific ATF3 ablations. We show that ATF3-KO mice exhibit a significantly reduced expression of cardiac remodeling markers following chronic pressure overload. Consistently, the lack of ATF3 specifically in either cardiomyocytes or BM derived cells blunts the hypertrophic response to PE infusion. A unique cross-talk between cardiomyocytes and macrophages was identified. Cardiomyocytes induce an ATF3 dependent induction of an inflammatory response leading to macrophage recruitment to the heart. Adoptive transfer of wild type macrophages, but not ATF3-KO derived macrophages, into wild type mice potentiates maladaptive response to PE infusion. Conclusions: Collectively, this study places ATF3 as a key regulator in promoting pressure overload induced cardiac hypertrophy through a cross-talk between cardiomyocytes and macrophages. Inhibiting this cross-talk may serve as a useful approach to blunt maladaptive remodeling processes in the heart. (C) 2015 Elsevier Ireland Ltd. All rights reserved.