关键词： 原发性肝癌   ATF3   MMP-2   maspin   肿瘤转移   组织芯片   免疫组织化学   EMSA   图像分析  

摘要： 目的:从蛋白水平上,检测肝细胞癌(hepatocellular carcinoma,HCC)组织芯片中ATF3、MMP-2及maspin的表达情况和手术切除新鲜肝癌标本中ATF3的DNA结合活性,探讨组织芯片中ATF3、MMP-2及maspin的表达与HCC侵袭转移的关系及其意义,及新鲜肝癌标本中ATF3的DNA结合活性与HCC侵袭转移的关系及其意义。 方法:对手工制作组织芯片的方法进行改良,构建含有HCC、癌旁肝组织、远癌肝组织、正常肝组织及肝内转移灶的组织芯片,应用免疫组化及彩色图像分析技术从蛋白质水平上检测了126例HCC、126例癌旁肝组织、81例远癌肝组织、20例正常肝组织和33例肝内转移灶中ATF3、MMP-2和maspin的表达情况,同时收集新鲜肝癌标本,利用凝胶电泳迁移率分析法( Electrophoretic Mobility Shift Assay, EMSA)检测其中的26例HCC、10例癌旁肝组织中ATF3的DNA结合活性情况,并综合分析ATF3、MMP-2和maspin的表达,ATF3的DNA结合活性与各临床病理参数的关系及其意义。 结果: 1、利用改良的手工制作组织芯片方法构建7个含有HCC、癌旁肝组织、远癌肝组织、正常肝组织以及肝内转移灶共185个点的芯片蜡块。其中中间列5个点为阳性对照点及标志点。所有蜡块点阵排列整齐,无裂纹,能完整切下。芯片经H-E、免疫组化染色后切片有效率达95%以上,芯片组织符合镜下组织学观察和进一步研究的需要。 2、免疫组织化学染色结果显示ATF3及MMP-2在HCC中表达高于癌旁肝组织、远癌肝组织和正常肝组织(P<0.01),maspin在HCC中的表达明显低于癌旁肝组织、远癌肝组织和正常肝组织(P<0.01)。 3、ATF3蛋白在转移组中表达高于无转移组(P<0.01),在女性HCC患者中的表达量高于男性患者(P<0.01)。此外,ATF3蛋白的表达与肿瘤大小、包膜侵犯、组织学类型以及病理学分级均无关(P>0.05)。MMP-2蛋白在包膜侵犯组中表达高于无包膜侵犯组(P<0.05),肝内转移灶表达量高于原发灶(P<0.05),在直径大于3cm的HCC中的表达量高于直径小于3cm的(P<0.05),但是MMP-2蛋白随HCC病理学分级的升高,相对表达量降低(P<0.05)。此外,MMP-2蛋白的表达与性别、组织学类型均无关(P>0.05)。ATF3随着HCC的转移进展表达量逐渐增高,MMP-2亦随着HCC的包膜侵袭、转移进展表达量逐渐增高,然而maspin却随着HCC病理学分级的升高,相对表达量降低(P<0.05)。此外,maspin蛋白的表达与性别、肿瘤大小、包膜侵犯、组织学类型以及转移情况均无关(P>0.05)。 4、ATF3蛋白表达与MMP-2表达呈正相关(P<0.05),与maspin表达呈负相关(P<0.01);MMP-2蛋白表达与maspin表达呈负相关(P<0.01)。 5、EMSA实验结果显示:ATF3蛋白在HCC中的DNA结合活性明显高于癌旁肝组织(P<0.01);在HCC组织中,转移组ATF3的DNA结合活性显著高于无转移组(P<0.05);此外,ATF3的DNA结合活性与HCC患者年龄、性别、组织学类型、肿瘤大小、包膜侵犯、病理学分级均无明显相关性(P>0.05)。 结论: 1、ATF3蛋白在转移组中表达高于无转移组,在女性HCC患者中的表达量高于男性患者。此外,ATF3蛋白表达与肿瘤大小、包膜侵犯、组织学类型以及病理学分级均无关。 2、MMP-2蛋白在包膜侵犯组中表达高于无包膜侵犯组,肝内转移灶表达量高于原发灶,在直径大于3cm的HCC中的表达量高于直径小于3cm的,但是MMP-2蛋白随HCC病理学分级的升高,相对表达量降低。此外,MMP-2蛋白的表达与性别、组织学类型以及转移情况均无关。ATF3及MMP-2均随着HCC进展表达量升高,然而maspin却随着HCC病理学分级的升高,相对表达量降低。此外,maspin蛋白的表达与性别、肿瘤大小、包膜侵犯、组织学类型以及转移情况均无关。 3、ATF3蛋白和MMP-2蛋白在肝癌组织中高表达,而maspin蛋白在肝癌组织中低表达。ATF3蛋白表达与MMP-2表达呈正相关,与maspin表达呈负相关;MMP-2蛋白表达与maspin表达呈负相关。在肝癌的侵袭转移过程中,ATF3和MMP-2、maspin之间存在相互调控机制。 4、在EMSA检测结果中,ATF3的DNA结合活性在HCC中高于癌旁肝组织,转移组ATF3的DNA结合活性高于无转移组。提示ATF3的DNA结合活性与HCC的侵袭转移

摘要： Overall, our studies are consistent with Atf3 acting directly on the IL6 promoter to inhibit further transcription of IL6 mRNA following stimulation with ET-1, given that Atf3 protein binds directly to the promoter (Fig. 5) and knockdown of the protein enhances IL6 mRNA expression (Fig. 4). Klf2 also appears to operate in a negative feedback system to limit IL6 mRNA expression since its knockdown also enhances IL6 mRNA expression (Fig. 6), although further studies are required to determine whether its effects are directly mediated at the level of the IL6 promoter. Interestingly, Ereg appears to be co-regulated with IL6 with a similar pattern of expression in response to ET-1 and similar enhancement of expression by cycloheximide (Fig. 2), Atf3 knockdown (Fig. 4) or Klf2 knockdown (Fig. 5). However, for both IL6 and Ereg, other factors may further contribute to the downregulation of mRNA expression following the increase induced by ET-1, possibly by increasing its rate of degradation. It is interesting that ET-1 also increased expression of Zfp36 (Table 1) which is associated with regulation of mRNA stability (Carrick et al., 2004). This and other factors may be required to limit the expression of IL6, Ereg and other selected IEGs responding to stimuli such as ET-1, and ensure that they are expressed only transiently. Understanding how these early changes associated with intercellular or intracellular signalling and gene/protein expression relate to the changing environment of a cell will help us understand how a complex phenotypic response such as cardiac hypertrophy may develop in vivo and how such an initially beneficial response may deteriorate into (for the heart) cardiac failure. © 2009 Elsevier Ltd. All rights reserved.

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： 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。

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： 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

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

摘要： Abstract: Endothelial cells (ECs) play an important role in hypoxia-induced vascular disorders. We investigated the acute hypoxia effect on 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% O2, pO2 =8mmHg) 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 a 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. [Copyright &y& Elsevier]

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。