关键词： Kinetics of IL-8 production and CagA status   Different CagA-type H. pylori strains (Western CagA-specific   sequence (WSS) and Eastern CagA-specific sequence (ESS)   Low pH condition   Activating transcription factor 3 (ATF3)  

摘要： We examined the kinetics of IL-8 production and CagA status in AGS infected with Helicobacter pylori, 26695 (Western CagA-type), HPK5 (Eastern CagA-type) and isogenic cagA-disrupted mutants, exposed to different pH (pH 6 and pH 3). IL-8 was produced in the early and Late phases after infection in CagA-dependent and independent manners, respectively, irrespective of CagA-type. The wild-type exposed to low pH tremendously reduced IL-8 level at early phase, but restored with urea, suggesting that low pH exerted the kinetics of H. pylori-induced IL-8 production in CagA-dependent manner and urea was necessary for effective induction. CagA and phosphorylated CagA increased time-dependently after infection. Phosphorylated CagA from 26695, but not HPK5, rapidly peaked, consistent with the kinetics of IL-8 induction and appearance of hummingbird phenotype. ATF3 transcripts peaked late phase by wild-type, however, induced in two peaks early and late phases by cagA-disrupted mutants, indicating that different CagA-type proteins altered ATF3 induction in the infected cells. (C) 2007 Elsevier GmbH. All rights reserved.

关键词： Apoptosis   Diabetes   Islet transplantation   Pancreatic beta-cells   Stress responses  

摘要： The focus of the research performed in this dissertation is on ATF3, a stress inducible transcription factor that plays a central role in the biology of β-cells. The hypothesis of this dissertation is that ATF3 functions by contributing to the expression of target genes that strongly influence β-cell survival and function. Previous studies demonstrated that activating transcription factor 3 (ATF3) is a stress-inducible gene and pro-apoptotic in various cell types, including β-cells. These results support a model that ATF3 plays a role in the pathogenesis of diabetes, partly by its apoptotic effect in β-cells. Based on this information, I set out to investigate the potential functional significance of ATF3 expression in β-cells exposed to two physiologically relevant stress paradigms; islet transplantation and high fat diet induced type 2 diabetes. In chapter 2, I tested whether ATF3 plays a role in the β-cell stress response to islet transplantation. In the course of this study, I compared wild type (WT) and ATF3 knockout (KO) islets in vitro using stress paradigms relevant to islet transplantation: isolation, inflammation, and hypoxia. I also compared the WT and KO islets in vivo using a syngeneic mouse transplantation model. As a result, I found that ATF3 was induced in all three stress paradigms and played a deleterious role in islet survival, as evidenced by the lower viability of WT islets than KO islets. ATF3 regulated various downstream target genes in a stress-dependent manner. These target genes can be classified into two functional groups: (a) apoptosis (NOXA, bNIP3), and (b) immuno-modulation (TNFα, IL1β, IL6, and CCL2/MCP-1). In vivo, ATF3 KO islets performed better than WT islets after transplantation, as evidenced by better glucose homeostasis in recipients, and reduced caspase 3 activation and macrophage infiltration in the KO grafts. These results identified a role for ATF3 in islet graft rejection by contributing to islet apoptosis and inflammatory

关键词： ATF3   融合蛋白   原核表达   ATF3   合成多肽   单克隆抗体  

摘要： 第一部分大鼠ATF3基因原核表达质粒的构建及其在大肠杆菌中的表达与纯化 目的:构建包含大鼠激活转录因子3(activating transcription factor 3,ATF3)基因不同长度片段的3个重组原核表达质粒,并在大肠杆菌中表达。方法:PCR扩增出大鼠ATF3基因及其截短片段,克隆至原核表达载体pGEX-4T-1,重组质粒pGEX-4T-1/ATF3, pGEX-4T-1/ATF3(129-546bp)和pGEX-4T-1/ATF3 (237-546bp)经酶切鉴定、核酸序列测定后转化大肠杆菌(***)BL21和rosetta,异丙基-β-D-硫代半乳糖苷(isopropylβ-D-thiogalactopyranoside,IPTG)诱导表达。通过SDS-PAGE,Western blot进行分析和鉴定,并且经过谷胱苷肽琼脂糖的层析柱层析纯化。结果:经酶切鉴定和核酸序列测定证实重组质粒构建正确。经过IPTG诱导以后,三个重组质粒在SDS-PAGE均未见到预期的目的条带,但是Western blot结果表明,质粒pGEX-4T-1/ATF3能在BL21中表达分子量为48KD的融合蛋白。融合蛋白用谷胱苷肽层析柱纯化,SDS-PAGE显示,纯化蛋白纯度较低。结论:成功构建了包含ATF3不同长度片段的3个重组原核表达质粒,其中pGEX-4T-1/ATF3能够在宿主菌BL21中正确表达。 第二部分抗大鼠ATF3单克隆抗体的制备与鉴定 目的:制备针对大鼠激活转录因子3(activating transcription factor 3,ATF3)蛋白的单克隆抗体。方法:根据软件合成偶联有匙孔绒血蓝蛋白(key hole limpet hemocyanin,KLH)的ATF3第168-181位氨基酸多肽,免疫Balb/c小鼠,体外将骨髓瘤细胞SP2/0和小鼠脾细胞融合,ELISA法筛选出阳性克隆,经有限稀释法以及三次亚克隆后筛选出能分泌抗ATF3蛋白第168-181位氨基酸多肽单克隆抗体的杂交瘤细胞,快速定性试纸法对抗体的类型进行鉴定,并且通过Western blot鉴定此单克隆抗体的特异性。结果:经过三次亚克隆后得到一株能稳定分泌抗大鼠ATF3蛋白的单克隆抗体。快速定性试纸法鉴定此单克隆抗体的亚型为IgG1,轻链为κ链。Western blot结果显示这株单抗能够特异性识别细胞内以及宿主菌BL21表达的ATF3蛋白。结论:成功制备了一株抗大鼠ATF3蛋白的单克隆抗体,为进一步研究ATF3的生物学功能提供了有用的实验工具。

关键词： 激活转录因子3(ATF3)   shRNA   肾小球系膜细胞   肾小球系膜细胞   亚溶解剂量的C5b-9复合物(sublytic C5b-9)   激活转录因子3(ATF3)   凋亡  

摘要： 目的:构建并鉴定大鼠野生型激活转录因子3(activating transcription factor 3, ATF3)基因及大鼠ATF3特异性小发夹RNA(small hairpin RNA, shRNA)真核表达质粒,并观察其在大鼠肾小球系膜细胞(glomerular mesangial cells, GMCs)中过表达及沉默ATF3基因的情况。方法:(1)用DNA重组技术构建大鼠ATF3基因真核表达质粒pcDNA3.1/ATF3和构建针对ATF3基因不同位点的五个shRNA序列的真核表达质粒ATF3 shRNA1-5。(2)体外分别将pcDNA3.1/ATF3质粒或ATF3 shRNA1-5质粒转染GMCs,筛选pcDNA3.1/ATF3和ATF3 shRNA转染最佳时间,并将GMCs作相应的分组处理。Western blot检测ATF3蛋白的相对表达量来鉴定pcDNA3.1/ATF3的表达及筛选最佳沉默效率shRNA。结果:(1)核酸序列测定表明,pcDNA3.1/ATF3质粒及ATF3 shRNA质粒构建正确。Western blot证实,pcDNA3.1/ATF3质粒成功表达ATF3蛋白,ATF3 shRNA-1具有最佳沉默效率。结论:(1)成功构建了大鼠野生型ATF3基因真核表达质粒pcDNA3.1/ATF3。(2)成功构建并筛选出最佳沉默效率的大鼠ATF3 shRNA-1。该结果为进一步研究ATF3基因在亚溶解型补体C5b-9(sublytic C5b-9)诱导GMCs凋亡病变中的作用奠定了基础。 目的:检查大鼠GMCs受sublytic C5b-9刺激后,其ATF3基因mRNA丰度和蛋白表达水平的变化,探讨过表达或靶向沉默ATF3基因对sublytic C5b-9诱导GMCs凋亡病变的影响。方法:(1)以sublytic C5b-9刺激GMCs为实验模型,应用RT-PCR、Real-time PCR、Western blot和免疫组化技术检查ATF3 mRNA丰度和蛋白表达水平,用Annexin V(AV)- propidium iodide (PI)双染后流式细胞仪定量分析GMCs凋亡数量。(2)体外将pcDNA3.1/ATF3质粒或ATF3 shRNA质粒瞬时转染GMCs,并将GMCs作相应的分组处理,再用Hoechst 33342染色检查各组GMCs的凋亡形态,并用AV-PI双染后流式细胞仪定量分析GMCs凋亡数量。结果:(1)Sublytic C5b-9刺激GMCs后,ATF3 mRNA及蛋白表达能迅速增高,至3h达到峰值;ATF3蛋白主要分布在GMCs细胞核及核周;Sublytic C5b-9刺激的GMCs,细胞凋亡率较对照组显著增高。(2)Sublytic C5b-9刺激的GMCs和转染pcDNA3.1/ATF3质粒的GMCs均呈现核Hoechst 33342染色致密增强或碎裂等凋亡形态学的改变;同时,GMCs凋亡百分率也明显增高。pcDNA3.1/ATF3质粒转染的GMCs再给予sublytic C5b-9刺激后,上述现象更为明显。与之相反,转染ATF3 shRNA质粒的GMCs再给予sublytic C5b-9刺激后,其GMCs凋亡率则显著降低。结论:Sublytic C5b-9刺激GMCs能诱导GMCs中ATF3表达上调和细胞凋亡,而ATF3表达增加对GMCs的凋亡有促进作用。

关键词： 尿道下裂   DEHP   MEHP   TGF-β1   ATF3  

摘要： 背景:尿道下裂(Hypospadias)是小儿最常见的泌尿生殖道先天畸形。由于阴茎及前尿道发育不全,尿道开口异常伴阴茎下弯畸形,导致患儿不能站立排尿,并影响成年后性功能及生育力。手术矫正畸形是本病的唯一治疗措施,但术后并发症发生率较高,是患儿及家长的心患。为此,有关尿道下裂病因的研究受到学者们关注,以期通过尿道下裂病因学的研究寻找其预防、治疗学突破。 尿道下裂发病率约1/200～1/300,而且有逐年增加趋势,大量研究提示其发病率的增加与环境雌激素污染密切相关。邻苯二甲酸二(2-乙基)己酯(DEHP)是一种广泛应用于塑料及化工产业的增塑剂,在医疗工作和日常生活中被大量应用,已构成我国严重的“白色污染”。DEHP进入环境后不易被降解,能蓄积于人体产生雌激素样生物活性。课题组前期研究已发现DEHP可损害实验动物睾丸及附睾功能导致小鼠隐睾畸形,可同时伴有尿道下裂的产生。虽有文献报道DEHP能诱发实验动物的尿道下裂,但是未就其机制深入研究。因此,本课题针对DEHP对外生殖器的毒性损伤,研究其致畸机制,为进一步防治尿道下裂发生提供科学依据。 目的:利用DEHP的外生殖器的毒性损伤,诱导胎鼠尿道下裂:体内外观察阴茎发育情况,检测细胞凋亡改变、TGF-β1及ATF3等在阴茎组织中的表达;对比尿道下裂患者与正常儿童包皮TGF-β1表达差异,深入探讨尿道下裂发生机制。 方法:1.将C57BL/6及KM孕鼠分为对照组与DEHP处理组,DEHP处理组分为100 mg/kg·d、200 mg/kg·d与500 mg/kg·d三个剂量组(DEHP

摘要： Stimulation of GnRH receptors enhances expression of activating transcription factor (ATF) 3 in a pituitary gonadotroph cell line. The signaling pathway requires elevated cytosolic Ca2+ levels and activation of ERK and c-Jun N-terminal protein kinase. The signaling cascade was blocked by overexpression of either MAPK phosphatase (MKP)-1 or MAPK phosphatase-5 that dephosphorylate nuclear ERK and c-Jun N-terminal protein kinase. In addition, ATF3 biosynthesis was impaired after lentiviral-mediated expression of a constitutively active mutant of calcineurin A. Thus, MKP-1, MKP-5, and calcineurin may function as shut-off devices for GnRH receptor signaling. Expression of dominant-negative mutants of early growth response protein (Egr)-1, cAMP response element binding protein ( CREB), and ATF2 blocked the biosynthesis of ATF3, indicating that these transcription factors connect the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the ATF3 gene. This view was corroborated by chromatin immunoprecipitation experiments revealing that Egr-1 and the phosphorylated forms of CREB and ATF2 bound to the 5'-upstream region of the ATF3 gene in buserelin-stimulated gonadotrophs. Together the data indicate that the ATF3 gene is a bona fide target gene of Egr-1, CREB, and ATF2 in gonadotrophs. Moreover, we show that in gonadotrophs ATF3 bound to its own promoter under physiological conditions. The analysis of a lentiviral-transmitted ATF3 promoter/luciferase reporter gene, embedded into the chromatin of the cells, revealed that ATF3 blocked the activity of its own promoter. We additionally identified the chromogranin B gene as bona fide target gene of ATF3 in gonadotrophs. (Endocrinology 149: 6311-6325, 2008)

关键词： Hypoxia   Activating transcription factor 3   Mitogen-activated protein kinase   Nitric oxide   Reactive oxygen species   Matrix metalloproteinase-2  

摘要： Endothelial cells (ECs) play an important role in hypoxia-induced vascular disorders. We investigated the acute hypoxia electron endothelial expression of activating transcription factor 3 (ATF3), a stress-inducible transcription factor playing significant roles in cellular responses to stress. Bovine aortic ECs were subjected to acute hypoxia (1%O-2, pO(2) = 8 mmHg) and ATF3 expression was examined. ECs exposed to hypoxia transiently induced ATF3 expression. A transient increase in the activation of C-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in ECs was observed: however. only ECs pretreated with a specific inhibitor to JNK Suppressed the hypoxia-induced ATF3 expression. ECs exposed to acute hypoxia transiently increased endothelial nitric oxide (eNOS) activity. Pre-treating ECs with,I specific inhibitor to eNOS (L-NAME) or PI3-kinase significantly inhibited the hypoxia-induced JNK activation and ATF3 expression. ATF3 induction has been shown to inhibit matrix metalloproteinase-2) (MMP-2) expression. Consistently, ECs exposed to hypoxia attenuated the MMP-2 expression. This hypoxia-attenuated MMP-2 expression can be rescued by pre-treating ECs with an inhibitor of eNOS. These results suggest that the ATF3 induction by acute hypoxia is mediated by nitric oxide and the JNK pathway in ECs. Our findings provide a Molecular basis for the mechanism in which ECs respond to acute hypoxia. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

关键词： 前列腺癌   激活转录因子3   免疫组织化学  

摘要： 目的:探讨激活转录因子3(ATF3)在前列腺癌的表达和意义。方法:采用免疫组化和WesternBlot检测正常前列腺、良性前列腺增生及前列腺癌组织中ATF3的表达。结果:ATF3在前列腺癌组织中的表达较正常前列腺组织和前列腺增生组织高(P<0.05)。ATF3的表达与临床分期、病理分级、是否伴有转移有关。结论:ATF3可能与前列腺癌的发生、发展、侵袭和转移有关,可以作为预测前列腺癌的恶性程度和预后的指标。

关键词： activating transcription factor 3   genetics   hypospadias   sex differentiation   urogenital abnormalities  

摘要： Purpose: ATF3, an estrogen responsive gene expressed during genital development, could be implicated in the etiology of hypospadias. ATF3 is up-regulated in the foreskin of patients with hypospadias and is implicated in suppression of the cell cycle, which may interfere with urethral cell growth. We sought to investigate the sequence of ATF3 in patients with hypospadias. Material and Methods: Direct sequencing of coding exons and splice sites of ATF3 was performed in 41 boys with hypospadias and 30 controls. In addition, ATF3 expression in 1 human fetal penis with and 1 without hypospadias was studied by immunohistochemical analysis. Results: A missense variant (L23M) was identified in a boy with anterior hypospadias. This amino acid is highly conserved. Three genomic variants (C53070T, C53632A, Ins53943A) were found in or close to exon 6 in patients with perineal, penoscrotal and anterior hypospadias. This important exon includes splice sites for an alternative transcript (ATF3 Delta Zip) that have been implicated in regulation of the function of ATF3. None of these genomic variants was present in controls. Immunochemical analysis revealed that in normal fetuses ATF3 is not expressed in and around the urethra, while in patients with hypospadias ATF3 is over expressed in the urethral plate and subcutaneous tissue, especially around the ectopic orifice of the urethra. Conclusions: Genomic variants of ATF3 are present in 10% of our patients with hypospadias. We also report an abnormal expression pattern of ATF3 in a hypospadiac fetus. The direct implication of ATF3 in the occurrence of hypospadias remains to be confirmed by functional studies of the genomic variants we describe.