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1.
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;MTT检测目的蛋白可有效地封闭TNFα对QSG7701的细胞毒性作用;流式细胞术检测目的蛋白对TNF-α诱导QSG7701凋亡有抑制作用;sTNFR1-MBP具有良好的生物活性,为今后的研究打下了基础。 相似文献
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目的探讨高迁移率族蛋白(high mobility group box,HMGB)1对大鼠滑膜细胞STAT信号通路的调控作用。方法将常规培养大鼠滑膜细胞株RSC-364细胞随机分为正常对照组和10μg/LHMGBI刺激组,分别培养6h、12h和24h,RT—PCR检测STAT1/3mRNA的表达,Western印迹法和流式细胞术(flow cytometric analysis,FCM)检测STAT1、STAT3、磷酸化STAT1(phospho—STAT1,p—STAT1)和磷酸化3(phospho—STAT3,p—STAT3)蛋白的表达,免疫细胞化学(ICC)检测PCNA蛋白的表达。结果HMGB1刺激6h~24h组STAT1 mRNA和p-STAT1蛋白的相对表达量呈时间依赖性上调,24h表达最高,与正常对照组相比差异均具有显著统计学意义(P〈0.05,P〈0.01)。PCNA蛋白阳性信号表达于细胞核内,呈棕黄色颗粒,且随着刺激时间延长阳性信号逐渐增强。RT—PCR、ICC染色和FCM均显示STAT3mR—NA和p-STAT3蛋白的相对表达量各组间相比差异均无统计学意义。结论HMGB1可能通过激活STAT1信号转导通路促进滑膜细胞的增殖和分化。 相似文献
3.
禽流感病毒HA基因真核表达质粒的构建与表达 总被引:15,自引:3,他引:15
血凝素蛋白(HA)基因是禽流感病毒(AIV)重要的保护性抗原基因.为了研究 HA基因疫苗,用PCR扩增H5亚型AIV HA基因,将其克隆到质粒pcDNA4/HisMax和pRc/CMV上得到真核表达质粒pC4H5和pCMVH5.采用TfxTM-20、Superfect转染试剂和电转染法转染HeLa细胞,转染后的HeLa细胞经蛋白质印迹和血凝试验检测HA蛋白及其活性.结果表明,Superfect转染和电转染均能正确表达HA蛋白并具有生物学活性,蛋白质印迹检测到HA和HA裂解的HA1和HA2,与AIV 的HA、HA1、HA2蛋白的分子质量一致.从血凝试验结果看,Superfect和电转染表达的HA均具有血凝活性,而经Superfect转染的pC4H5的表达量是pCMVH5的8倍,表明pC4H5是一高效的真核表达质粒. 相似文献
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TALF(Tachyleus antilipoposaccharide factor)对细菌内毒素(LPS)的核心部分有抑制作用。研究TALF cDNA基因在大肠杆菌中的表达,首先将TALF cDNA基因分别插入大肠杆菌表达载体pGEX-4T-2、pET22b、pET28a中,构建重组表达质粒,转化于大肠杆菌BL21(DE3)。结果表明克隆于pET22b、pET28a中的TALF cDNA基因没有表达,而融合了GST的TALF基因(GST-TALF)能够在大肠杆菌中表达,并形成包涵体。从1L培养基中可获得4mg纯度为91%的GST-TALF融合蛋白。经复性和纯化后的融合蛋白GST-TALF几乎检测不到抑菌活性及LPS中和活性,但该融合蛋白经凝血酶消化后表现出明显的体外抑菌活性及LPS中和活性。 相似文献
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该实验以小鼠系膜细胞MMC为研究对象,以重组HMGB1为刺激物,通过检测细胞周期的变化及细胞PCNA、CyclinD1、CDK4和p16的表达水平,初步探讨HMGB1对系膜细胞的细胞周期及其相关调控因子的影响。选取小鼠系膜细胞MMC为研究对象,随机分为对照组及0.05mg/LHMGB1刺激组,经流式细胞术检测发现HMGB1能够上调小鼠系膜细胞中S期细胞所占比例;免疫细胞化学检测显示,PCNA蛋白在小鼠系膜细胞中的表达上调;通过RT-PCR技术及Western blot技术检测到小鼠系膜细胞中CyclinD1 mRNA和蛋白以及CDK4蛋白的高表达情况,而p16蛋白的表达呈时间依赖性降低。由此可见,HMGB1可能是通过上调CyclinD1/CDK4的表达,并下调p16的表达,促进细胞从G_0/G_1期进入S期,介导了小鼠系膜细胞的异常增殖,可能是HMGB1参与狼疮性肾炎发病的可能机制之一。 相似文献
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人骨形成蛋白3在昆虫杆状病毒表达系统中的表达、纯化及其诱骨活性检测 总被引:3,自引:0,他引:3
为了在昆虫杆状病毒系统中表达人骨形成蛋白 3(humanbonemorphogeneticproteoin3,hBMP3) ,检测表达产物的诱骨活性 .将hBMP3全长cDNA 1416bp克隆入转移载体K8中 ,再与病毒DNA经脂质体包裹后转染昆虫细胞Sf9.重组病毒经蓝白筛选后 ,用PCR方法扩增目的基因片段进行初步鉴定 .收集重组病毒的转染上清 ,肝素亲和层析纯化目的蛋白 .SDS PAGE和Western印迹进一步鉴定此蛋白 .体外培养MC3T3 E1细胞 ,经目的蛋白刺激后 ,检测细胞内碱性磷酸酶 (ALP)活性 .并将目的蛋白进行小鼠体内肌肉包埋实验 ,检测其异位诱骨活性 .结果显示 ,肝素亲和层析可以收集 1个高的洗脱峰 .SDS PAGE显示 ,非还原型样本为 32kD的二聚体蛋白带和少量 16kD单体蛋白带 ;还原型样本仅有相应大小的单体蛋白带 ,纯度达 80 % .Western印迹在相应位置呈阳性染色 .此蛋白刺激小鼠成纤维细胞 4 8h后 ,胞内ALP的活性增高 ,经rhBMP3作用后的细胞MTT染色减弱 .在小鼠股部肌肉内包埋蛋白样品 2~ 3周 ,组织学检测有成骨组织生成 .说明hBMP3在此套系统中获得了表达 .表达产物在体外可以刺激成骨细胞的分化 ,却抑制细胞的增殖 .小鼠体内异位诱骨实验进一步证实表达产物具有成骨活性 相似文献
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摘要 目的:探讨长链非编码核糖核酸(LncRNA)KCNQ1OT1靶向调控miR-124-3p/高迁移率族蛋白B1(HMGB1)轴对高糖诱导肾小球系膜细胞(HMC)增殖、凋亡及纤维化的影响。方法:将人HMC分为对照组(NC组)、高糖组(30 mmol/L葡萄糖)、阴性序列(si-NC)组、KCNQ1OT1小干扰核糖核酸(RNA)(si-KCNQ1OT1)组、si-KCNQ1OT1+模拟对照序列(miR-NC)组、si-KCNQ1OT1+miR-124-3p抑制剂(miR-124-3p inhibitor)组,各组在转染后进行高糖处理。实时荧光定量聚合酶链式反应(RT-qPCR)检测LncRNA KCNQ1OT1信使核糖核酸(mRNA)、miR-124-3p mRNA、HMGB1 mRNA表达;四甲基偶氮唑盐比色法(MTT)检测细胞增殖活性;流式细胞术检测细胞凋亡率;蛋白印迹法(Western blot)检测HMGB1蛋白、增殖相关蛋白[细胞周期蛋白1(CyclinD1)]、细胞凋亡蛋白[半胱氨酸蛋白酶-3(caspase-3)、半胱氨酸蛋白酶蛋白9(caspase-9)]、细胞纤维化蛋白[纤维连接蛋白(FN)、细胞黏附分子-1(ICAM-1)]表达;双荧光素酶报告基因实验验证LncRNA KCNQ1OT1与miR-124-3p与HMGB1之间的靶向关系。结果:与NC组比较,高糖组KCNQ1OT1 mRNA、HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性(24 h、48 h和72 h)明显上升,miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显下降(P<0.05);与高糖组、si-NC组比较,si-KCNQ1OT1组KCNQ1OT1 mRNA、HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性(24 h、48 h和72 h)明显下降,miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显上升(P<0.05);与si-KCNQ1OT1组、si-KCNQ1OT1+miR-NC组比较,si-KCNQ1OT1+miR-124-3p inhibitor组HMGB1 mRNA及HMGB1蛋白、CyclinD1蛋白、FN蛋白、ICAM-1蛋白表达、HMC活性(24 h、48 h和72 h)明显上升,miR-124-3p mRNA、caspase-3蛋白及caspase-9蛋白表达、HMC凋亡率明显下降(P<0.05)。较miR-NC组与KCNQ1OT1-WT共转染组而言,miR-124-3p mimic组与KCNQ1OT1-WT共转染组细胞荧光素酶活性明显降低(P<0.05);较miR-NC组与HMGB1-WT共转染组而言,miR-124-3p mimic组与HMGB1-WT共转染组细胞荧光素酶活性明显降低(P<0.05)。结论:LncRNA KCNQ1OT1可以靶向下调miR-124-3p mRNA表达,上调HMGB1 mRNA及HMGB1蛋白表达,促进高糖诱导HMC增殖,抑制凋亡,促进细胞纤维化发展。 相似文献
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目的:探讨EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节,为EGb761的临床运用提供可行的依据。方法:LPS(1μg/m L)诱导不同时间后,western blotting检测THP-1细胞上清液中HMGB1蛋白含量变化及不同浓度EGb761对LPS诱导THP-1细胞释放HMGB1蛋白的表达和NF-κB的活性;酶联免疫吸附法(ELISA)检测细胞中IL-1β、IL-6、TNF-α的含量。共聚焦显微镜观察EGb761对LPS诱导THP-1细胞释放HMGB1蛋白核转位变化。结果:(1)LPS组IL-1β、IL-6、TNF-α的含量在刺激6-12 h后明显高于空白对照组,而EGb761+LPS组IL-1β、IL-6、TNF-α的含量均显著低于LPS组(P0.05)。(2)EGb761处理LPS诱导THP-1细胞6 h后细胞上清液NF-κB活性表达量较空白对照组低,随着处理时间延长至12 h,NF-κB的活性表达量呈明显下降趋势(P0.05)。(3)LPS诱导THP-1细胞18 h后,细胞上清液中HMGB1蛋白含量呈明显升高趋势(P0.05)。(4)不同浓度EGb761对LPS诱导THP-1细胞18 h后,HMGB1蛋白含量较空白对照组有下降趋势,HMGB1蛋白含量随着EGB761浓度增加至100μg/m L呈下降趋势并呈浓度依赖效应(P0.05)。(5)LPS诱导THP-1细胞后,在共聚焦显微镜下可见胞浆中大量HMGB1蛋白标记分布,而EGb761+LPS共同诱导THP-1细胞后胞浆中可见少量HMGB1蛋白分布。结论:LPS可诱导THP-1细胞IL-1β、IL-6、TNF-α表达增多及NF-κB活化,导致HMGB1蛋白表达增多及核转位,而EGB761能抑制THP-1细胞IL-1β、IL-6、TNF-α表达及NF-κB活化,调节HMGB1蛋白的表达及核转位。 相似文献
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目的:通过观察高迁移率族蛋白1(HMGB1)、转录因子NF-E2相关因子2(Nrf2)及血红素加氧酶1(HO-1)基因沉默对白血病化疗耐药细胞(K562/A02细胞株)的影响,探讨该信号通路在白血病化疗耐药中的作用及其可能机制。方法:将HMGB1基因、Nrf2基因及HO-1基因的特异性干扰RNA分别转染阿霉素耐药细胞株K562/A02,荧光实时定量(RT-PCR)方法检测HMGB1、Nrf2及HO-1的mRNA表达水平,Western blot方法检测HMGB1、Nrf2及HO-1的蛋白表达水平,免疫荧光方法检测Nrf2的蛋白表达,并使用CCK-8方法检测转染前后K562/A02细胞株的细胞活性。结果:HMGB1基因、Nrf2基因或HO-1基因沉默的K562/A02细胞活性皆显著低于对照组及空白组(P0.05),化疗敏感性恢复。结论:HMGB1高表达导致了白血病细胞株K562/A02对阿霉素的化疗耐药,Nrf2/HO-1信号通路参与了HMGB1诱导的K562/A02细胞的化疗耐药,其表达上调可恢复K562/A02细胞对阿霉素的敏感性。 相似文献
11.
Xiaojin Wu Yanyan Mi Hui Yang Ankang Hu Qingguo Zhang Chunli Shang 《Molecular and cellular biochemistry》2013,380(1-2):249-257
Extracellular high-mobility group box-1 (HMGB-1) has been implicated in the inflammation response leading to the precancerous lesions of non-small cell lung cancer (NSCLC). However, the role of HMGB-1 in the inflammation response in normal human bronchial epithelial (NHBE) cells and its underlying mechanisms were still not fully understood. In this study, the inflammation response in NHBE cells was stimulated by 2.5, 5, and 10 μg/ml HMGB-1. However, the receptor for advanced glycation end products (RAGE) blocker RAGE-Ab (5 μg/ml) or 10 μM c-Jun N-terminal kinases (JNK) inhibitor SP600125 could inhibit HMGB1-induced the release of inflammation cytokines including TNF-α, IL-8, IL-10, and MCP-1 in a dose-dependent manner. Furthermore, HMGB1-induced RAGE protein expression, JNK and NF-κB activation were attenuated by the pretreatment with RAGE-Ab or JNK inhibitor SP600125 in Western blot analysis. Our data indicated that HMGB-1 induced inflammation response in NHBE cells through activating RAGE/JNK/NF-κB pathway. HMGB-1 could act as a therapeutic target for inflammation leading NHBE cells to the precancerous lesions of NSCLC. 相似文献
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Hu YM Pai MH Yeh CL Hou YC Yeh SL 《American journal of physiology. Renal physiology》2012,302(1):F150-F158
Acute kidney injury (AKI) is a severe complication of sepsis. High-mobility group box (HMGB)-1 was implicated as a late mediator of lethal systemic inflammation in sepsis. Since glutamine (GLN) was shown to have anti-inflammatory and antioxidant properties, we hypothesized that GLN administration may downregulate an HMGB-1-mediated pathway and thus ameliorate sepsis-induced AKI. Mice were randomly assigned to a normal group (NC), a septic saline group (SS), or a septic GLN group (SG). Sepsis was induced by cecal ligation and puncture (CLP). The SS group was injected with saline, and the SG group was given 0.75 g GLN/kg body wt once via a tail vein 1 h after CLP. Mice were killed 2, 6, and 24 h after CLP, and blood and kidneys of the animals were harvested for further analysis. The results showed that sepsis resulted in higher mRNA and/or protein expressions of kidney HMGB-1, toll-like receptor (TLR) 4, myeloid differentiation primary-response protein (MyD) 88, and receptor of advanced glycation end products (RAGE) compared with normal mice. Septic mice with GLN administration exhibited decreased HMGB-1, TLR4, RAGE, and phosphorylated NF-κB p65 protein expressions and reduced nitrotyrosine levels in kidney tissues. The histological findings showed that damage to the kidneys was less severe, and survival improved in the SG group. These results indicated that a single dose of GLN administered after the initiation of sepsis plays a prophylactic role in downregulating the expressions of HMGB-1-related mediators and decreasing oxidative stress in the kidneys, which may consequently have ameliorated AKI induced by sepsis. 相似文献
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Davis K Banerjee S Friggeri A Bell C Abraham E Zerfaoui M 《Molecular medicine (Cambridge, Mass.)》2012,18(1):359-369
Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation. 相似文献
18.
Hofner P 《Acta microbiologica et immunologica Hungarica》2007,54(4):323-337
High mobility group box 1 protein (HMGB-1), a nuclear protein is a critical cytokine that mediates the response to infection, injury and inflammation. The aim of our study was to elaborate a reliable in vitro model to investigate whether Mycobacterium bovis BCG is able to induce HMGB-1 secretion from the monocytic U-937 cells. Western blot technique was applied for the detection of HMGB-1 from supernatants of cells, following induction with LPS, Staphylococcus aureus, and Mycobacterium bovis BCG. HMGB-1 was subjected to MALDI-TOF mass and PSD analysis. Quantitation of the secreted HMGB-1 was performed by ELISA. The BCG strain induced higher amounts of secreted HMGB-1 than LPS or Staphylococcus aureus. The translocation of the HMGB-1 to the cytoplasm following infection of cells with BCG was demonstrated by immunofluorescence examinations. CONCLUSION: Our pilot experiments draw attention the to HMGB-1-inducing ability of Mycobacterium bovis. Assessment of the pathophysiological role of this late cytokine in mycobacterial infections demands further in vitro and in vivo examinations. 相似文献
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Hyperlipidemia stimulates the extracellular release of the nuclear high mobility group box 1 protein
Our aim was to evaluate the effect of hyperlipidemia on the activation of endogenous alarmin, the high mobility group box
1 (HMGB1) protein, related to systemic inflammation associated with the progression of experimental atherosclerosis and to
establish whether statin treatment regulates the HMGB1 signaling pathway. Hyperlipidemia was induced in vivo in golden Syrian
hamsters and in monocyte cell culture (U937) by feeding the animals with a high-fat Western diet and by exposing the cells
to hyperlipidemic serum. Blood samples, heart, lung and cells were harvested for biochemical, morphological, Western blot,
quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses. The data revealed that, in the atherosclerotic
animal model, the protein HMGB1 and its gene expression were increased and that fluvastatin treatment significantly reduced
the release of HMGB1 into the extracellular space. The cell culture experiments demonstrated the relocation of HMGB1 protein
from the nucleus to cytoplasm under hyperlipidemic stress. The high level of detected HMGB1 correlated positively with the
up-regulation of the advanced glycation end product receptors (RAGE) in the lung tissue from hyperlipidemic animals. During
hyperlipidemic stress, the AKT signaling pathway could be activated by HMGB1-RAGE interaction. These results support the existence
of a direct correlation between experimentally induced hyperlipidemia and the extracellular release of HMGB1 protein; this
might be controlled by statin treatment. Moreover, the data suggest new potentials for statin therapy, with improved effects
on patients with systemic inflammation induced by hyperlipidemia. 相似文献
20.
Jee Seung Han Hyun Ah Kim Sanghyun Lee Minhyung Lee 《Journal of cellular biochemistry》2010,110(5):1094-1100
High mobility group box‐1 (HMGB‐1) is a nuclear protein that can bind to and condense plasmid DNA. In this study, we developed a recombinant VEGF receptor binding peptide (VRBP) linked to HMGB‐1 box A (VRBP‐HMGB1A) as a targeting gene carrier to hypoxic endothelial cells. Hypoxic endothelial cells in ischemic tissues of solid tumors are important targets for gene therapy. A recombinant VRBP‐HMGB1A expression vector, pET21a‐VRBP‐HMGB1A was constructed. VRBP‐HMGB1A was over‐expressed in BL21 strain and purified by nickel‐chelate affinity chromatography. Complex formation between VRBP‐HMGB1A and pCMV‐Luc was confirmed by gel retardation assay. pCMV‐Luc was retarded completely at a 2/1 weight ratio (peptide/plasmid). For transfection assays, calf pulmonary artery endothelial (CPAE) cells were incubated under hypoxia for 24 h, prior to transfection to induce the VEGF receptors on the cells. VRBP‐HMGB1A/pCMV‐Luc complexes were transfected to hypoxic CPAE cells. The highest transfection efficiency was at a 30/1 weight ratio (peptide/plasmid). In addition, VRBP‐HMGB1A had higher efficiency than poly‐L ‐lysine (PLL) specifically in hypoxic CPAE cells, However, VRBP‐HMGB1A had lower efficiency than PLL in 293, H9C2, and normoxic CPAE cells. In MTT assay, VRBP‐HMGB1A was less toxic than PLL to cells. In conclusion, VRBP‐HMGB1A is a potential gene carrier for targeting hypoxic endothelial cells and thus, may be useful for cancer gene therapy. J. Cell. Biochem. 110: 1094–1100, 2010. Published 2010 Wiley‐Liss, Inc. 相似文献