人乳头瘤病毒16E6基因通过增加HMGB1表达调节口腔癌CAL27细胞侵袭的实验研究

人乳头瘤病毒16型(Human papillomavirus type 16,HPV16)感染与口腔癌、宫颈癌的发病有关,HPV16 E6基因编码的蛋白是重要的癌蛋白,已经被证实能够通过增加高迁移率族蛋白B1(High mobility group box-B1,HMGB1)表达来促进宫颈癌细胞的侵袭,但是否能调控口腔癌细胞的侵袭仍未明确。为研究HPV16 E6基因通过增加HMGB1表达调节口腔癌CAL27细胞侵袭的作用,口腔癌CAL27细胞被分为对照组、空白质粒组、HPV16 E6质粒组、NC-si RNA组(短片断干扰RNA阴性对照组)、NC-si RNA+HPV16 E6质粒组、HMGB1-si RNA+HPV16E6质粒组,检测细胞中HPV16 E6及HMGB1的表达、细胞的侵袭数目、培养基中HMGB1的含量。结果显示,HPV16 E6质粒组细胞中HPV16 E6及HMGB1的表达量、培养基中HMBG1的含量、细胞的侵袭数目均高于对照组及空白质粒组(P<0.05);HMGB1-si RNA组细胞中HMGB1的表达量明显低于对照组及NC-si RNA组(P<0.05);NC-si RNA+HPV16 E6质粒组的细胞侵袭数目均明显高于NC-si RNA组(P<0.05),HMGB1-si RNA+HPV16 E6质粒组的细胞侵袭数目均明显低于NC-si RNA+HPV16 E6质粒组(P<0.05)。本研究提示,HPV16 E6基因能够促进口腔癌CAL27细胞的侵袭且这一作用与增加HMGB1表达有关。     

人HMGB1分子的克隆、重组蛋白表达与生物学活性

采用RT-PCR,从重症肝炎病人外周血单个核细胞（peripheral blood mononuclear cells PBMCs）的mRNA中扩增高迁移率族蛋白1（high mobility group box－1 protein,HMGB1）基因,构建重组表达质粒pGEX4T-1-HMGB1,转化入大肠杆菌,测序证实其序列与基因数据库中HMGB1基因(NM_002128)一致。诱导表达的融合蛋白GST-HMGB1,用Glutathione Sepharose 4B亲和层析纯化,经Thrombin酶切得到HMGB1。结果显示：经Western-blotting检测,GST-HMGB1 和HMGB1均具有免疫活性; ELISA和MTT检测发现,两者均能刺激RAW264.7细胞产生大量TNF-α,明显刺激HeLa细胞增殖。GST-HMGB1具有良好的生物学活性,为今后的研究打下了基础。     

HMGB1与冠心病关系的研究进展

高迁移率族蛋白B1(high mobility group box1protein,HMGB1)是一种高度保守的核内非组蛋白,可由致病因素引发细胞被动释放或损伤、凋亡和坏死细胞主动分泌而产生。HMGB1可以在真核细胞坏死过程中被释放到细胞外。越来越多的研究证明,HMGB1是一种重要的潜在炎性介质,通过与其特异性受体结合而发挥着重要的炎症细胞因子作用。近年来研究表明,HMGB1通过参与炎症过程而影响动脉粥样硬化斑块的进展。因此,HMGB1可能是机体冠状动脉炎性疾病的重要靶点。本文对HMGB1及其作用机制进行综述,为心血管疾病的预防和治疗提供文献依据。     

HMGB1在脑缺血再灌注损伤过程中的作用及其相关抑制剂的研究进展

高迁移率族蛋白1 (high mobility group box-1, HMGB1)是一种含有215个氨基酸残基的核蛋白,几乎存在于所有真核细胞中,参与调节染色体结构、基因转录、DNA复制和修复等多项生理过程,在生长发育过程中发挥着重要作用。越来越多的研究结果显示,HMGB1通过多种途径参与细胞凋亡、自噬和炎症反应等多种病理生理过程,从而诱导脑缺血再灌注(cerebral ischemia-reperfusion, CIR)损伤。此外,目前发现存在多种物质能靶向抑制HMGB1的表达,进而发挥抗炎、抗细胞凋亡和抗氧化损伤等作用,但具体作用机制仍需更加深入的研究。进一步挖掘和探索HMGB1在CIR损伤中的具体作用及其相关抑制剂将为CIR损伤的临床治疗提供新的潜在候选策略和思路。因此,本文就HMGB1在CIR损伤过程中的作用及其相关抑制剂的研究进展作一简要综述。     

HMGB1在抗肿瘤免疫发生中的作用

高迁移率族蛋白B1(high mobility group box 1, HMGB1)是一种广泛分布于哺乳动物细胞的高度保守的核蛋白。HMGB1对肿瘤的发生发展具有一定的影响,能够通过多种信号分子途径来促进肿瘤的增殖、迁移和侵袭等。随着相关研究的不断深入,人们发现,HMGB1与免疫细胞抗肿瘤作用的发挥也有密切联系。因此,HMGB1有望成为肿瘤免疫治疗的重要靶点之一。然而,HMGB1生物学功能复杂,对肿瘤免疫的影响作用也不单一。为了更好地将HMGB1应用于肿瘤治疗,本文就HMGB1在抗肿瘤免疫发生中的作用及其机制的研究进展作一综述。     

低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响*

目的:探讨低氧时人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC)的高迁移率族蛋白1(HMGB1)及相关受体和炎症因子表达,并检测HMGB1对两种细胞增殖、迁移活性的影响。方法:低氧(1%氧浓度,Hypoxia组)及常氧(Control组)条件下培养HPASMC和HPAEC,RealTime-PCR检测两种细胞HMGB1、TLR2、TLR4、TLR9、RAGE、CD24、IL-6 、TNF-a和CXCL8 mRNA等受体和炎性因子的表达。MTS法观察不同浓度HMGB1对HPASMC和HPAEC增殖的影响;划痕法观察HMGB1对HPASMC和HPAEC迁移的影响。结果:Hypoxia组HPASMC、HPAEC中HMGB1及RAGE mRNA表达量较Control 组明显升高(P＜0.05及0.01);Hypoxia组HPAEC中CD24及HPASMC中IL-6 mRNA表达明显增高(P均＜0.05)。MTS结果显示在345 pmol/L 剂量下 HMGB1明显抑制HPAEC的增殖(P＜0.01),而对HPASMC增殖无影响。划痕实验示HMGB1对HPASMC和HPAEC迁移无明显影响。结论:低氧诱导HPAEC、HPASMC 产生HMGB1;HMGB1通过抑制HPAEC增殖引起内皮屏障功能障碍;而低氧进一步刺激HPASMC产生炎症因子。     

HMGB1蛋白在病毒感染致病机制中的作用研究进展

高迁移率族蛋白1(HMGB1)是一种高度保守的核蛋白,广泛存在于哺乳动物细胞内,在核内参与核小体的构建与稳定以及基因的转录,核外参与介导炎症反应。近年多项研究表明,HMGB1参与了多种病毒(SARS、HIV、HPV、HSV等)感染的致病过程,本文就其研究进展作一综述。     

不同潮气量机械通气对大鼠肺组织HMGB1表达的影响

目的观察不同潮气量机械通气大鼠肺组织高迁移率族蛋白1（HMGB1） mRNA及其蛋白的表达水平,探讨HMGB1在呼吸机相关性肺损伤（VILI）发病中的作用。方法24只雄性Wister大鼠随机分为对照组、小潮气量组和大潮气量组,分别采用原位分子杂交技术和免疫组织化学染色法检测肺组织HMGB1 mRNA及其蛋白的表达水平。结果与对照组和小潮气量组比较,大潮气量组大鼠肺组织HMGB1 mRNA及其蛋白表达水平明显升高（均P〈0.01）,小潮气量组与对照组各项指标比较差异无统计学意义（P〉0.05）。结论大潮气量机械通气可诱导肺组织HMGB1 mRNA及其蛋白高表达;HMGB1分泌增多导致肺组织炎症反应扩大,可能是呼吸机相关性肺损伤（VILI）发生的重要因素之一。     

人端粒结合蛋白TRF1的克隆、表达和抗体制备

利用RT-PCR技术,从HeLa细胞的cDNA文库中扩增到人端粒结合蛋白1(hTRF1)基因编码区序列,克隆至pUCm-T载体,测序正确后,构建带His₆-tag原核表达载体pET-28c-TRF1,经IPTG诱导表达的His₆-TRF1融合蛋白分子量约为65kD,Western-blot证实表达产物可特异地与TRF1抗体sc-6165结合。用Ni^2＋NTA胶亲和层析纯化可得到电泳均一的融合蛋白,免疫新西兰纯种大白兔,获得特异性好的多克隆抗体,该抗体可用于免疫荧光染色和Western-blot方法检测哺乳动物细胞内源性的TRF1分子。     

小鼠HMGB1启动子荧光素酶报告基因的构建及功能鉴定

利用PCR技术扩增小鼠高迁移率族蛋白1(HMGB1)基因启动子序列,构建小鼠HMGB1启动子荧光素酶报告基因pGL3-basic-HMGB1.经PCR、酶切及测序鉴定后,用脂质体法将pGL3-basic-HMGB1转入巨噬细胞264.7中,并应用萤光素酶测定系统检测其活性.检测结果显示pGL3-basic-HMGB1具有启动子活性.小鼠HMGB1启动子荧光素酶报告基因pGL3-basic-HMGB1的成功构建,为进一步研究HMGB1提供基本材料.     

人sTNFR1基因原核表达及体外抗肝细胞凋亡的活性研究

采用RT-PCR,从Hela细胞的mRNA中扩增人sTNFR1基因,构建含有目的片段的T载体克隆及原核表达载体pMAL-c2x重组质粒亚克隆,转化入大肠杆菌,测序证实其序列与基因数据库中sTNFR1基因一致.经异丙基-β-D半乳糖苷酶（IPTG）诱导表达,淀粉树脂亲和层析法纯化,得到融合蛋白sTNFR1-MBP.结果显示：经Western-blotting检测,sTNFR1-MBP具有免疫活性L流式细胞术检测目的蛋白对TNF-α诱导QSG7701凋亡有抑制作用,这为今后的研究打下了基础.     

HIV感染者/AIDS病人高迁移率族蛋白1的表达及其临床意义

通过检测74例处于不同病期的HIV感染者/AIDS患者和10例健康对照者PBMCs中HMGB1 mRNA的表达水平及其外周血浆HMGB1、TNF-a和IL-2水平,比较各组之间表达水平的差异及其与CD4＋T淋巴细胞的关系.发现HMGB1 mRNA的表达水平及血浆HMGB1含量在AIDS病人组明显高于感染者组和正常对照组（P〈0.05）;AIDS患者经HAART治疗后疗效差组HMGB1 mRNA的表达水平及血浆HMGB1含量也明显高于疗效好组（P〈0.05）;而经HAART治疗后效果好且免疫功能恢复的患者HMGB1 mRNA的表达及血浆HMGB1含量均较治疗前明显下降（P〈0.05）;当CD4＋T细胞数低于200/μL时,血浆HMGB1含量以及PBMCs中HMGB1 mRNA表达水平与CD4＋T细胞数呈负相关.显示HMGB1在HIV/AIDS发病及病情进展过程中可能起重要作用,HMGB1血浆含量及PBMCs中HMGB1 mRNA的表达水平高低与HIV/AIDS患者病情轻重密切相关.     

MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis

Atherosclerosis has been recognized as a chronic inflammatory disease, which can harden the vessel wall and narrow the arteries. MicroRNAs exhibit crucial roles in various diseases including atherosclerosis. However, so far, the role of miR-328 in atherosclerosis remains barely explored. Therefore, our study concentrated on the potential role of miR-328 in vascular endothelial cell injury during atherosclerosis. In our current study, we observed that oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis and inhibited cell viability dose-dependently and time-dependently. In addition, indicated dosage of ox-LDL obviously triggered HUVECs inflammation and oxidative stress process. Then, it was found that miR-328 in HUVECs was reduced by ox-LDL. HUVECs apoptosis was greatly repressed and cell survival was significantly upregulated by overexpression of miR-328. Furthermore, mimics of miR-328 rescued cell inflammation and oxidative stress process induced by ox-LDL. Oppositely, inhibitors of miR-328 strongly promoted ox-LDL-induced endothelial cells injury in HUVECs. By using bioinformatics analysis, high-mobility group box-1 (HMGB1) was predicted as a downstream target of miR-328. HMGB1 has been reported to be involved in atherosclerosis development. The correlation between miR-328 and HMGB1 was validated in our current study. Taken these together, it was implied that miR-328 ameliorated ox-LDL-induced endothelial cells injury through targeting HMGB1 in atherosclerosis.     

The interaction of HMGB1 and linker histones occurs through their acidic and basic tails

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.     

A comparison of HMGB1 concentrations between cerebrospinal fluid and blood in patients with neurological disease

Aims: To determine whether a correlation exists between paired cerebrospinal fluid (CSF) and serum levels of a novel inflammatory biomarker, high-mobility group box 1 (HMGB1), in different neurological conditions.

Methods: HMGB1 was measured in the serum and CSF of 46 neurological patients (18 idiopathic intracranial hypertension [IIH], 18 neurological infection/inflammation [NII] and 10 Rasmussen’s encephalitis [RE]).

Results: Mean serum (±?SD) HMGB1 levels were 1.43?±?0.54, 25.28?±?27.9 and 1.89?±?1.49?ng/ml for the patients with IIH, NII and RE, respectively. Corresponding mean (±?SD) CSF levels were 0.35?±?0.22, 4.48?±?6.56 and 2.24?±?2.35?ng/ml. Both CSF and serum HMGB1 was elevated in NII. Elevated CSF HMGB1 was demonstrated in RE. There was no direct correlation between CSF and serum levels of HMGB1.

Conclusion: Serum HMGB1 cannot be used as a surrogate measure for CSF levels. CSF HMGB1 was elevated in NII and RE, its role as a prognostic/stratification biomarker needs further study.     

重组人半乳凝集素-1的原核表达、纯化及生物活性检测

目的:在大肠杆菌中表达半乳凝集素-1(galectin-1),并进行纯化及生物活性检测。方法:将人半乳凝集素-1基因克隆至带有His融合标签的原核表达载体pQE-30上,转化大肠杆菌M15,经IPTG诱导表达,表达产物经亲和层析纯化后,进行Western印迹鉴定,并用红细胞凝集试验检测其生物学活性。结果:双酶切鉴定和核苷酸序列测定表明重组表达质粒pQE-30-Galectin-1构建正确;重组蛋白的表达量约占菌体总蛋白的50%,主要以可溶形式表达,纯化后蛋白纯度达95%以上,且具有良好的红细胞凝集活性。结论:在大肠杆菌中表达了重组人半乳凝集素-1,且具有良好的生物活性。     

人HMGB1 B Box的表达及其生物活性

经RTPCR从人新鲜扁桃体组织中扩增人高迁移率族蛋白B1(HMGB1)中的Bbox(88～162残基)的cDNA,构建于载体pUC19,经测序后与GenBank中报道的已知序列完全一致,再构建于原核表达载体pQE80LDHFR中,表达并鉴定目的蛋白.经Ni2+亲和层析柱、多粘菌素B层析柱纯化获得高纯度的DHFRHMGB1Bbox蛋白,然后将此重组HMGB1Bbox加入到人外周血单核细胞(PBMCs)中,37℃,5%CO2下,刺激PBMCs6h,用ELISA检测PBMCs释放TNFα、IL6的量,经检测后证明纯化后的重组HMGB1Bbox能显著地刺激PBMCs释放致炎因子TNFα、IL6.HMGB1Bbox的表达及其生物活性的初步研究,为进一步研究HMGB1Bbox的作用机制以及新型抗炎制剂的研发奠定基础.     

LPS诱导肝细胞L02释放HMGB1蛋白的研究

以人单核巨噬细胞系U937细胞为对照,探讨肝细胞L02分泌晚期炎症介质高迁移率族蛋白HMGB1过程的特点.400μg/L LPS作用于人U937细胞和L02细胞,MTT和荧光TUNEL结果显示0～24 h,两种细胞均未见明显坏死和凋亡.RT-PCR结果表明L02细胞HMGB1 mRNA表达水平高于相应对照组的时相(20 h)晚于U937细胞(16 h).Western blot显示L02细胞上清中检测到HMGB1蛋白的时相(16 h)晚于U937细胞(8h);两者上清HMGBl蛋白水平呈时间依赖性,但U937细胞分泌HMGB1蛋白的量占有绝对优势.细胞免疫荧光观察到两者均有HMGB1从细胞核向胞浆移位的现象,但L02细胞晚于U937细胞.由此可见,LPS诱导肝细胞分泌晚期炎症介质HMGB1具有时相晚、量少的特点,且其分泌HMGB1的过程与HMGB1蛋白移位有关,而并不依赖于细胞坏死与凋亡.     

JH-4 reduces HMGB1-mediated septic responses and improves survival rate in septic mice

Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as attractive therapeutic strategies for the management of severe vascular inflammatory diseases. Recently, we found that JH-4, a synthesized decursin derivative, exhibited a strong anti-Hutchinson-Gilford progeria syndrome by efficiently blocking progerin-lamin A/C binding. In this study, we examined the effects of JH-4 on HMGB1-mediated septic responses and the survival rate in a mouse sepsis model. The anti-inflammatory activities of JH-4 were monitored based on its effects on lipopolysaccharide- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of JH-4 were determined by measuring permeability, leukocyte adhesion, migration, and the activation of proinflammatory proteins in HMGB1-activated human umbilical vein endothelial cells and mice. JH-4 inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. JH-4 also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with JH-4 reduced CLP-induced release of HMGB1, sepsis-related mortality, and pulmonary injury in vivo. Our results indicate that JH-4 is a possible therapeutic agent to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.