血管生成素相互作用蛋白质的筛选与鉴定

血管生成素（angiogenin, ANG）在肿瘤、神经退行性疾病和先天免疫过程中均发挥作用,但对其具体生理病理功能和作用机制的了解并不深入全面.蛋白质 蛋白质相互作用调控着细胞内的各个生物学过程,可以为目标蛋白质功能和机制的探索提供信息.本文利用酵母双杂交技术,分别从人心肌和肝cDNA文库中筛选了ANG的可能相互作用蛋白质.对筛选获得的20个候选蛋白质进行生物信息学分析,显示10个蛋白质含有EGF结构域;有5个蛋白质在KEGG 数据库已有记录,主要参与细胞黏附、通讯和迁移等过程.在以往的研究中,我们已经验证α 辅肌动蛋白2（α-actinin 2）、卵泡抑素（follistatin）、磷脂混杂酶1（phospholipid scramblase 1）和腓骨蛋白1（fibulin1）与ANG作用的真实性.本文的蛋白质沉降实验显示,ANG与腓骨蛋白2、3、4之间也存在相互作用.     

血管生长素促血管生成机制及其与疾病关系的研究进展

血管生长素(angiogenin,ANG)是一种促使新血管生成的诱导剂,它属于核糖核酸酶超家族中的一员。ANG蛋白含123个氨基酸,相对分子量为14．4kD,在肿瘤细胞中首先发现,也存在于正常细胞中,可由多种细胞分泌,其核糖核酸酶活性较弱。研究证实,ANG可以在细胞间及细胞内发挥促血管生长作用。ANG与缺血性疾病、肿瘤、及其它疾病的关系已见不少报道。本文简述ANG促血管生成作用机制及其与疾病的关系。     

血管生成素基因组结合序列的筛选与鉴定

血管生成素（angiogenin,ANG）调控细胞增殖、迁移、分化等生物学过程,但其作用的分子机制尚未完全明了. 目前认为,ANG可结合到rDNA区域促进rRNA转录,也可能与mRNA有结合.为全面鉴定细胞内可结合ANG的基因组序列,我们利用染色质免疫共沉淀结合DNA芯片技术（ChIP-chip）对HeLa细胞的基因组DNA进行了筛选,共获得了1 248个结合片段. 我们进一步分析了这些结合片段附近分布的基因,发现有699个可能受ANG结合调控的基因. 基因注释和聚类分析显示,这些可能受ANG调控的基因主要与肿瘤发生发展有关（特别是结直肠癌和前列腺癌）,并且与TGF-β和Wnt信号通路相关. 最后,我们验证了ANG不仅与WNT6、CCNE1、APC2、FZD8和EGFR基因的启动子区域有直接结合,而且调控其表达.以上研究结果为深入研究ANG的功能机制提供了线索.     

核糖核酸酶抑制因子对血管生成素功能的调控

血管生成素（angiogenin,ANG）能有效促进血管生成和肿瘤细胞增殖,在肿瘤发生发展中起重要作用.其主要分子机制是通过核转位和激活PI3K/AKT/mTOR信号通路,刺激rRNA转录和核糖体生成.ANG也被发现在肌萎缩侧索硬化症（ALS）和帕金森病（PD）患者中存在基因编码区的功能突变,表明其在运动神经元生理方面发挥作用,其缺陷是神经退行性疾病的一个危险因素.核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞内酸性蛋白质,由460个氨基酸残基组成,分子质量约为50 kD,当其与核糖核酸酶A（RNaseA）结合形成复合物后,可抑制RNaseA 的90％以上活性,从而有效调节细胞内RNA水平. ANG具有低核糖核酸酶活性, 是RNase超家族一员,与RNase A有着高度保守的同源顺序. 序列、结构和酶学等分析表明,RI也能够与ANG紧密结合,且得到体外实验的证明. 研究发现,RI具抑癌基因功能;RI与ANG在细胞内共定位;Co IP和GST pull down证实其相互作用,获取了RI与ANG在体内结合的直接证据;RI与AKT磷酸化表达负相关.在膀胱癌细胞及临床标本中证实了RI与 ANG和PI3K/AKT通路分子表达的相关性及与肿瘤细胞生长与转移的关系．在细胞和动物模型研究表明,RI调节ANG活性的功能及其分子机制,即RI通过结合ANG而封锁其核转位和调控PI3K/AKT/mTOR信号通路及其相关通路交互应答（cross talk）的能力,从而抑制肿瘤生长及转移. RI是一个有希望的抗肿瘤蛋白新药和血管生成抑制剂,可望成为基因治疗的靶基因.     

血管生成素的结构与功能的研究进展

血管生成素(angiogenin,ANG)是一种有效的血管生成因子,是RNase超家族中惟一具有促血管生成能力的成员,也是目前已知的所有血管生成因子中独具核糖核酸酶活性的因子。ANG具有3个功能元件,即RNase活性中心、细胞表面结合位点及核定位序列。ANG参与血管生成的各个阶段,是其他血管生成因子诱导新血管生成的枢纽,其作用受到受体调节。在肿瘤的发生、发展及恶化过程中,ANG也具有非常重要的作用。通过对ANG促血管生成及细胞增殖机制的研究,为治疗肿瘤提供了多种靶点和途径。     

血管生成素专刊序言

血管生成素（angiogenin）是1985年发现的.为展示30年来血管生成素相关领域研究进展,加强对血管生成素功能、作用机制及其与疾病关系的认识和理解,《中国生物化学与分子生物学报》在第7届编辑委员会成立之际委托我组织专刊.本期专刊包括9篇综述、4篇研究报告及1篇技术与方法方面的论文. 肿瘤血管生成是在上世纪70年代初由哈佛医学院附属波士顿儿童医院外科医生Jodan Folkman在临床实践中发现并提出的概念.为获得刺激肿瘤血管生成的因子,Folkman与哈佛医学院的生物化学家Bert Vallee教授合作,经过10年努力终于在1985年从肿瘤细胞培养液中分离得到具有强力促进血管生成能力的蛋白质--血管生成素.经过30年的研究,目前认为血管生成素不仅可促进肿瘤血管生成,它还可根据细胞类型和/或所处环境不同,或促进细胞的增殖、迁移、粘附能力,或保护细胞免受细胞应激的损伤,从而在发育、先天免疫等生理过程和肿瘤、神经退行性疾病等病理过程中发挥作用. 本专刊的14篇文章均是结合各研究组的工作经历和专长而组织的,涵盖了国内从事血管生成素研究的主要单位,并邀请了美国和意大利的两家单位撰写综述.浙江大学许正平教授研究组的 “血管生成素研究的希望与挑战--浙江大学血管生成素研究”一文不仅总结了他们14年来从蛋白质相互作用、基因转录调控、RNA代谢等层面研究血管生成素功能机制的工作进展,而且分析了该领域研究存在的问题和可能的发展方向.军事医学科学院郑晓飞教授研究组在国际上首先建立了血管生成素与tRNA片段产生的关系;“血管生成素介导的tRNA片段化”不仅回顾了其发现过程,而且总结了血管生成素介导产生的tRNA片段的功能和作用机制,并分析了其在临床上的可能应用.重庆医学大学陈俊霞教授研究组长期从事核糖核酸酶抑制因子（Ribonuclease inhibitor,RI）对血管生成素功能的影响研究,“核糖核酸酶抑制因子对血管生成素功能的调控”不仅阐述了RI的结构与功能,而且结合其研究进展总结了RI调节血管生成素促血管生成和细胞增殖活性的机制.军事医学科学院徐东刚教授研究组鉴定了FHL3是血管生成素的相互作用蛋白,结合其研究工作和同行报道撰写的“血管生成素在炎症性疾病中的研究进展”讨论了血管生成素在炎症性疾病特别是肿瘤中的作用和调控机制,提出它可能是兼具抗炎和抗血管生成双重功能的作用靶点.胡国富教授曾经在Vallee实验室领导血管生成素细胞生物学功能研究,做出了一系列创新性工作.在本期专刊中,他的研究组贡献了3篇综述.“血管生成素在细胞凋亡调节中的作用机制”阐述了其作为一种抗凋亡因子的作用途径,包括对内源性 和外源性凋亡信号分子的调控.“血管生成素在前列腺癌中的功能和作用机制”首先分析了前列腺癌的研究现状和临床治疗方案,指出转移性去势抵抗前列腺癌是目前治疗的难点,而血管生成素促进的rRNA转录不仅是前列腺上皮内瘤样病变所必需的,而且是前列腺癌细胞雄激素不依赖生长所必需的,可能是良好的治疗靶点.“血管生成素在造血系统恶性肿瘤中的功能和作用机制”是一篇探讨性的综述,在指出血液恶性肿瘤这类非实体肿瘤也是血管生成依赖疾病的基础上,作者陈述了大量事实以证明血管生成素与白血病、骨髓增生异常综合征、恶性髓系血液病等相关,但其在疾病中的作用及机制不清,需要深入研究.意大利那不勒斯菲里德里克第二大学的Daria Maria Monti在血管生成素和载脂蛋白A I（apolipoprotein A I,ApoA I）两个领域均有原创性的工作;“血管生成素在淀粉样蛋白病中发挥作用吗？”一文概述了血管生成素在淀粉样蛋白病中的研究进展,分析了血管生成素失调与该类疾病起始及进展间的关系.华中科技大学同济医学院的吴云霞在与胡国富合作开展新霉胺治疗宫颈癌、前列腺癌研究的基础上,开展新霉胺治疗肿瘤的临床前研究,其撰写的“基于血管生成素的药物研发展望”分析了该蛋白质在功能上的特殊性及其药物研发潜力. 浙江大学自2001年建立血管生成素研究组后,先后进行了血管生成素相互作用蛋白质、基因组结合序列、相关miRNA的筛选工作,获得了一批基础数据.“血管生成素相互作用蛋白质的筛选与鉴定”、“血管生成素基因组结合序列的筛选与鉴定”、“靶向调控血管生成素miRNA的筛选和验证”报道了这些基础数据,希望有助于推动我国血管生成素的研究工作.东北师范大学王丽教授在“新霉胺通过抑制血管生成素活性抑制人黑色素瘤细胞增殖、迁移和侵润”一文中报道了他们的最新研究结果. 虽然在1987年就实现了血管生成素在大肠杆菌中的高表达和分离纯化,但随后不断有技术和方法学上的改良,没有进行系统的总结.“重组人血管生成素制备和生物活性鉴定”总结了浙江大学血管生成素研究组多年来在该蛋白质的表达、纯化、活性分析方面的经验. 尽管血管生成素的研究已经有30年的历史,但对其功能机制的了解仍不够全面和深入.个人认为,其主要原因是血管生成素在小鼠体内有5个同源基因和3个假基因,可能存在基因功能的重复和冗余;在人体细胞内,虽然只有单拷贝的血管生成素基因,但与同一家族的核糖核酸酶4受同一启动子控制,功能上也存在重复和冗余.因此,传统的基因敲除技术难以揭示血管生成素的功能.同时,除在星形胶质细胞上鉴定多配体蛋白聚糖4（syndecan 4）是介导血管生成素选择性内吞的受体外,其它细胞上的受体至今没有明确报道,也影响了对其功能机制的研究.随着科学技术的发展,特别是CRISPR基因编辑技术的出现和优化,同时敲除多个基因已成为可能,将有力推动血管生成素的研究.我们相信,在不久的将来,血管生成素的功能机制研究将取得突破性进展.组织本期专刊的目的一方面是纪念血管生成素发现30周年,另一方面是向读者介绍血管生成素的研究现状、存在问题和可能的突破方向,以期启发大家的思路,共同为血管生成素功能机制研究贡献力量.     

蛋白质酪氨酸磷酸酶与药物靶标

蛋白质酪氨酸磷酸化作用是真核细胞中的一种重要信号作用机制,由蛋白质酪氨酸激酶和蛋白质酪氨酸磷酸酶共同调控.蛋白质酪氨酸磷酸酶在真核细胞代谢进程中起着重要的作用,与许多人类疾病如肿瘤、心血管疾病、免疫缺陷性疾病、传染病、神经性以及代谢方面疾病的发病机制密切相关,许多蛋白质酪氨酸磷酸酶已成为研究和开发治疗人类重大疾病药物的优秀靶标.     

血管生长素与治疗性血管生成的研究进展

血管生长素(angiogenin,ANG) 是一种分泌性的单链碱性蛋白质,由123个氨基酸组成,分子量为14.4kD,广泛分布在人体中.ANG属于核糖核酸酶超家族中的一员,具有低核糖核酸酶活性.研究证实,ANG是一种有效的促血管生成因子,参与血管生成的各个阶段,是其它血管生成因子诱导新血管生成的枢纽,在缺血性疾病的治疗方面显示出巨大的潜能.本文对ANG在治疗性血管生成方面的研究进展作一综述.     

猪ANG4在小肠表达的特征及细胞类型

血管生成素4(ANG4)是一种新型的抗菌肽,主要在哺乳动物的肠道组织表达,具有抗菌和促血管生成等功能,在机体对病原体的免疫过程和血管生成过程中发挥着重要的作用。鉴于该蛋白具有多种功能,ANG4在新生断奶仔猪肠道疾病防治方面具有非常广阔的前景。为了了解ANG4在仔猪肠道的表达特点,我们通过蛋白质免疫印迹、荧光定量PCR和免疫组化分析及其在肠道表达的细胞定位,分别确定了仔猪肠道内ANG4的mRNA、蛋白质表达水平以及在肠道表达的细胞定位。结果显示,ANG4的mRNA和蛋白质在仔猪肠道不同部位的变化趋势大致相同,除mRNA水平在断奶后有一个明显下降外,mRNA和蛋白质表达水平呈现随着周龄的增加而升高的趋势。ANG4蛋白的表达基本都集中于小肠绒毛,腺窝处也有少量表达,主要的表达细胞为肠上皮细胞以及潘氏细胞。     

趋化因子CXCL1在肿瘤中的作用

趋化因子及其受体在许多生物学过程(如炎症发生、血管生成等)中起重要的作用。趋化因子CXCL1是单亚基趋药性细胞因子,该蛋白主要通过特异性结合G蛋白耦联受体CXCR2,在多种肿瘤的生长、增殖、转移和侵袭以及血管新生中起重要调控作用。该文重点阐述了趋化因子CXC亚家族成员CXCL1在肿瘤中的功能,并对其上游调控因子进行分析,深入探讨了CXCL1与肿瘤的相互关系。     

基于血管生成素的药物研发

血管生成素（angiogenin,ANG）在伤口愈合、月经周期、妊娠、胚胎发育、先天性免疫、细胞应激保护和维持机体稳态等生理病理过程,特别是肿瘤的生存与进展、神经细胞的存活和生长中扮演着重要角色,是药物研发的重要靶点.本文综述了ANG在功能上的特殊性及其药物研发潜力.在肿瘤中,ANG扮演促进肿瘤细胞增生和促进血管生成的双重角色,且是其它血管生成因子如血管内皮生长因子（vascular endothelial growth factor, VEGF）、酸性成纤维生长因子（acidic fibroblast growth factor, aFGF）、碱性成纤维生长因子（basic fibroblast growth factor, bFGF）和表皮生长因子（epidermal growth factor, EGF）发挥作用的必需准许因子. ANG的抗肿瘤治疗较之目前常用的针对单一血管生成因子的抑制剂更有效,具有良好的药物研发和临床运用前景.由于ANG通过核转位促进rRNA转录是发挥促进肿瘤细胞增生和血管生成活性所必须的,因此,它的核转位抑制剂如新霉胺,将有望首先获得抗肿瘤临床应用.另外,业已证明重组ANG能促进体内外运动神经元的存活,且可明显改善肌萎缩侧索硬化症（amyotrophic lateral sclerosi, ALS）模型小鼠的行为,其在神经退行性疾病治疗方面也将有良好的研发前景.     

Anti‐inflammatory effects of doxepin hydrochloride against LPS‐induced C6‐glioma cell inflammatory reaction by PI3K‐mediated Akt signaling

Recent studies have shown that tricyclic antidepressants (TCAs) may have anti‐inflammatory and anticonvulsant effects in addition to its antidepressant effects. So far, the nonantidepressant effects of TCAs and their molecular pharmacological mechanisms remain completely unclear. Chronic inflammation in the brain parenchyma may be related to the pathogenesis and progression of various neurodegenerative diseases. As a common antidepressant and anti‐insomnia drug, doxepin also may be a potential anti‐inflammatory and anticonvulsant drug, so the study on the anti‐inflammatory protective effect of doxepin and its molecular mechanism has become a very important issue in pharmacology and clinical medicine. Further elucidating the anti‐inflammatory and neuroprotective effects of doxepin and its molecular mechanism may provide the important theoretical and clinical basis for the prevention and treatment of neurodegenerative disease. This study was designed to understand the glio‐protective mechanism of doxepin against the inflammatory damage induced by lipopolysaccharide (LPS) exposure in C6‐glioma cells. We found the treatment of C6‐glioma cells with LPS results in deleterious effects, including the augmentation of inflammatory cytokine levels (tumor necrosis factor‐α, interleukin‐1β), and suppresses the Akt phosphorylation. Furthermore, our outcomes demonstrated that doxepin was able to suppress these effects induced by LPS, through activation of the phosphatidylinositol‐3‐kinase‐mediated protein kinase B (Akt) pathway. To sum up, these results highlight the potential role of doxepin against neuroinflammatory‐related disease in the brain.     

Prolonged infusion of angiotensin II into normal rats induces stellate cell activation and proinflammatory events in liver

Recent evidence indicates that angiotensin II (ANG II) plays an important role in liver fibrogenesis. However, the underlying mechanisms are largely unknown. In advanced chronic liver diseases, circulating levels of ANG II are frequently elevated. We investigated the hepatic effects of prolonged systemic infusion of ANG II in normal rats. Saline or ANG II at subpressor and pressor doses (15 and 50 ng.kg-1.min-1, respectively) were infused to normal rats for 4 wk through a subcutaneous osmotic pump. Infusion of ANG II resulted in liver injury, as assessed by elevated serum liver enzymes. Livers from ANG II-perfused rats showed activation of JNK and ERK as well as increased NF-kappaB and activating protein-1 DNA-binding activity. Moreover, ANG II perfusion induced oxidative stress, increased concentration of proinflammatory cytokines, and upregulated the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2. Histological examination of the livers from ANG II-infused rats showed mild portal inflammation as well as thickening and thrombosis of small hepatic vessels. ANG II-treated livers showed accumulation of CD43-positive inflammatory cells and activated hepatic stellate cells (HSCs) at the pericentral areas. A slight increase in collagen synthesis was observed, as assessed by Sirius red staining and hepatic hydroxyproline. All of these effects were observed when ANG II was perfused at subpressor and pressor doses. ANG II also accelerated the activation of primary cultured rat HSCs. In conclusion, increased systemic ANG II can induce liver injury by promoting proinflammatory events and vascular damage. ANG II-induced hepatic effects are not dependent on increase in arterial pressure.     

Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT9‐mediated uric acid uptake

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti‐inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA‐induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP‐1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor‐kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose‐dependent manner. In contrast, phloretin significantly attenuated pro‐inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor‐kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA‐induced endothelial injury via a synergic mechanism including direct anti‐inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia‐related cardiovascular diseases.     

青蒿素类化合物与肥胖诱导的代谢性炎症

青蒿素类化合物是治疗疟疾的首选药物,具有高效、速效、低毒和安全的特点。近三十年来,大量研究结果证明青蒿素类化合物具有抗炎和免疫调节功能;这些研究主要集中于自身免疫性疾病,如类风湿性关节炎、全身性红斑狼疮、哮喘病和过敏反应等。最新研究表明,青蒿素类化合物可通过促进白色脂肪棕色化和增强棕色脂肪功能,起到预防肥胖的作用。作为肥胖过程中免疫细胞和炎症状况变化显著的脂肪组织,青蒿素类化合物是否参与其中的免疫炎症调节以及在该过程中发挥的潜在作用有待进一步实验的验证。本文对已有的青蒿素类化合物在抗炎和免疫调节方面的报道进行总结,并对青蒿素类化合物在改善肥胖诱导代谢性炎症方面的潜在应用进行展望。     

Essential role of PKC-zeta in normal and angiotensin II-accelerated neointimal growth after vascular injury

The contribution of atypical protein kinase C (PKC)-zeta to ANG II-accelerated restenosis after endoluminal vascular injury was investigated by using the rat carotid balloon injury model. Exposure of injured arteries to ANG II resulted in an extensive neointimal thickening (1.9 times) compared with vehicle at day 14. Treatment with PKC-zeta antisense, but not scrambled, oligonucleotides reduced neointimal formation observed in the presence or absence of ANG II. Examination of early events (2 days) after injury showed an increase in cellularity in the perivascular area of the artery wall that was transferred to the adventitia and media after exposure to ANG II, events blocked by PKC-zeta antisense, but not scrambled, oligonucleotides. A positive correlation between medial cellularity at day 2 and extent of neointimal growth at day 14 was established. Immunohistochemical analysis showed that upregulation of inflammatory markers after injury, as well as infiltration of ED1(+) monocytes/macrophages from the perivascular area to the adventitia, was accelerated by ANG II. However, ANG II-stimulated medial increase in cellularity was proliferation independent, and these cells were monocyte chemoattractant protein-1(+)/vimentin(+) but ED1(-)/VCAM(-). PKC-zeta is degraded after injury, and inhibition of its neosynthesis in medial vascular smooth muscle cells or in infiltrating cells with PKC-zeta antisense attenuated medial cellularity and expression of inflammation mediators without reversing smooth muscle cell dedifferentiation. Together, these data indicate that PKC-zeta plays a critical role in normal and ANG II-accelerated neointimal growth through a mechanism involving upregulation of inflammatory mediators, leading to cell infiltration in the media of the vascular wall.     

New soluble angiopoietin analog of Hepta‐ANG1 prevents pathological vascular leakage

Vascular leak is a key driver of organ injury in diseases, and strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the angiopoietin‐1 (ANG1)‐Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANG1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANG1 with a heptameric scaffold derived from the C‐terminus of serum complement protein C4‐binding protein α. We refer to this new fusion protein biologic as Hepta‐ANG1, which forms a stable heptamer and induces Tie2 phosphorylation in cultured cells, and in the lung following intravenous injection of mice. Injection of Hepta‐ANG1 ameliorates vascular endothelial growth factor‐ and lipopolysaccharide‐induced vascular leakage, in keeping with the known functions of Angpt1‐Tie2 in maintaining quiescent vascular stability. The new Hepta‐ANG1 fusion is easy to produce and displays remarkable stability with high multimericity that can potently activate Tie2. It could be a new candidate ANG1 mimetic therapy for treatments of inflammatory vascular leak, such as acute respiratory distress syndrome and sepsis.