HIV-1Nef调节内皮细胞黏附分子ICAM-1的表达

目的研究HIV-1的调节基因Nef对ECV304细胞ICAM-1表达的影响,从而为分析HIV-1感染引起内皮细胞生物学活性的变化,以及为阐明Nef参与HIV-1致病的分子机制奠定基础。方法应用本实验室已经建立保存的HIV-1Nef基因在内皮细胞的稳定表达细胞株ECV304-Nef和其阴性对照细胞株ECV304 pcDNA 3.1(+),通过RT-PCR、实时定量PCR(real-time PCR)、Western blot、FCM和细胞黏附试验分析ECV304-Nef细胞ICAM-1的表达水平。结果 RT-PCR、real-time PCR结果显示ECV304-Nef细胞ICAM-1 mRNA表达水平明显升高,为对照组的(4.3±0.2)倍;Western blot结果示ECV304-Nef细胞I-CAM-1蛋白的表达水平高于对照组;FCM分析显示ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P0.01),两组间ICAM-1表达有显著差异。细胞黏附实验观察到ECV304-Nef细胞黏附的Jurkat细胞数明显多于对照组,荧光仪定量分析结果显示ECV304-Nef细胞黏附的Jurkat细胞的荧光强度值显著高与对照组(P0.05)。结论本实验证实了HIV-1Nef基因可以上调血管内皮细胞细胞黏附分子ICAM-1的表达。     

HIV-1 Nef基因在THP-1细胞的表达和活性检测

目的将HIV-1 Nef基因转染THP-1细胞,获得稳定表达Nef蛋白的细胞克隆,为研究Nef对巨噬细胞生物学活性的影响奠定实验基础。方法将质粒pcDNA3.1(+)-Nef和pcDNA3.1(+()阴性对照)转染THP-1细胞,通过逆转录-聚合酶链反应(RT-PCR)、Western blot、细胞免疫荧光等方法检测目的蛋白在真核细胞内的表达及定位,采用共转染法将荧光报告基因转染THP-1Nef和THP-13.1细胞,通过测定荧光(RLU)值来评价Nef蛋白的生物学活性。结果转染细胞经G418筛选后获得稳定表达Nef的THP-1细胞株,RT-PCR及Western blot结果表明Nef在真核细胞中成功表达,细胞免疫荧光结果显示,THP-1-Nef细胞表达的Nef蛋白主要定位于细胞质中。荧光酶标仪检测转染了HIV-1LTR-Luc和NFκB-Luc荧光报告基因的THP-1-Nef和THP-1-3.1细胞的RLU值。结论成功建立了THP-1-Nef细胞稳定表达细胞株,检测了其生物学活性,为进一步研究其作用机制实验奠定基础。     

α-黑色素细胞刺激素对ECV304细胞与U937细胞黏附作用的影响

研究α-黑色素细胞刺激素(α-MSH)对人脐静脉内皮细胞系ECV304细胞黏附作用的影响,以探讨α-MSH的抗炎与免疫调节机制。以U937细胞与ECV304细胞相互黏附作为实验模型,观察LPS或TNF-α刺激ECV304细胞,以及α-MSH干预后,对U937细胞黏附于ECV304细胞的影响。用不同浓度α-MSH处理ECV304细胞,或在LPS/TNF-α处理的同时加不同浓度的α-MSH培养ECV304细胞,以FACS检测ICAM-1,RT-PCR检测骨桥蛋白(OPN)mRNA,Westernblot检测OPN蛋白表达。结果显示,LPS或TNF-α处理ECV304细胞可增加ECV304细胞与U937细胞的黏附,而α-MSH降低这种黏附作用。α-MSH抑制ECV304细胞表达ICAM-1和OPN蛋白;LPS或TNF-α刺激后,ECV304细胞表达OPN mRNA上调,而α-MSH能抑制LPS或TNF-α的上调作用。这些结果提示,α-MSH可通过降低ICAM-1和OPN的表达抑制炎症时血管内皮细胞与白细胞的黏附。     

ERK信号转导途径在LPS诱导的人类内皮细胞iNOS表达中的作用

目的主要研究 p44/42MAPK(ERK1/2或ERK)信号途径在LPS诱导的人内皮细胞 -ECV304iNOS表达和NO产生中的作用。方法用Griess法检测细胞培养液中NO水平 ,分别用免疫荧光法和RT -PCR检测细胞iNOS蛋白和mRNA表达 ,采用免疫激酶沉淀以Westernblot检测细胞中ERK激酶的活性。结果LPS可显著激活人类内皮细胞 -ECV304中的ERK信号途径 ,其激活高峰在LPS刺激后15～20min ,45min后活性逐渐下降。用ERK的特异性抑制剂PD98059处理细胞后可以显著抑制LPS诱导人内皮细胞ERK的活性。与对照组相比 ,LPS刺激后的ECV304细胞iNOS蛋白和mRNA的表达明显增加 ,培养基中NO水平也显著升高 ,用PD98059预处理细胞后 ,可显著抑制细胞iNOS表达和NO和产生。研究结果表明ERK信号途径参与了LPS介导的人内皮细胞iNOS表达和NO产生。结论通过阻断信号转导通路来减少iNOS及其它细胞因子的产生 ,为败血症休克的防治提供了一条新的思路     

血管内皮细胞(ECV304)表达的HLA-G1对NK细胞杀伤活性的抑制作用

目的:证实HLA-G1分子能够抑制NK细胞对同种血管内皮细胞系的杀伤作用。方法:采用脂质体介导的DNA转染技术,以构建的真核表达质粒pcDNA3-HLA-G1转染人脐静脉内皮细胞系ECV304,再用免疫荧光法检测表达的HLA-G1分子。并用MTT比色法检测HLA-G1对NK细胞杀伤活性的影响。结果:ECV304细胞上可稳定表达HLA-G1。NK细胞对空质粒pcDNA3转染的ECV304细胞的杀伤率为(50.6±18.1)%;而对pcDNA3-HLA-G1转染的ECV304细胞的杀伤率为(29.7±11.4)%,两者差异具有显著意义(P<0.01)。结论:HLA-G1分子可明显抑制NK细胞对同种血管内皮细胞的杀伤效应。     

海洋放线菌素X2下调柯萨奇病毒B3感染ECV304细胞ICAM-1的表达

目的:观察海洋放线菌素X2对柯萨奇病毒B3(CVB3)感染ECV304细胞细胞间黏附分子1(ICAM-1)表达的影响。方法:采用MTT法检测不同浓度的海洋放线菌素X2作用病毒后的抑制情况;采用RT-PCR和流式细胞术分别测定CVB3感染的ECV304细胞在海洋放线菌素X2作用前后不同时间点的ICAM-1的mRNA和蛋白的表达水平。结果:CVB3感染ECV304细胞后,ICAM-1蛋白表达在12 h～54 h显著高于正常ECV304细胞对照组(P<0.05);海洋放线菌素X2干预后,于12 h～54 h降低ICAM-1蛋白的表达,与CVB3感染组相比差异有统计学意义(P<0.05);海洋放线菌素X2干预后,于24 h～54 h降低ICAM-1mRNA的表达,与CVB3感染组相比差异有统计学意义(P<0.05)。结论:海洋放线菌素X2在病毒吸附前对CVB3具有抗病毒作用,并可下调CVB3诱导的ECV304细胞ICAM-1的mRNA和蛋白的表达。     

反式脂肪酸对ECV304细胞HNP-1mRNA表达和氧自由基的作用

目的:探讨反式脂肪酸(TFAs)对ECV304细胞氧化低密度脂蛋白(ox-LDL)的作用及其机制。方法:按常规方法传代培养ECV304细胞,用实时荧光定量PCR检测不同浓度TEAs刺激(TFA处理组)后ECV304细胞中HNP-1的表达;采用流式细胞仪检测ECV304细胞中氧自由基(ROS)的生成;用脂质氧化产物丙二醛(MDA)生成量描述ECV304细胞LDL氧化能力的变化。并与未行TFAs处理的ECV304细胞对照组比较其组间差异。结果:与对照组比较,TFAS处理组HNP-1mRNA表达水平显著升高(0.220±0.030vs 0.429±0.090,P0.05);与对照组比较,1mmol/L TFAS刺激ECV304细胞12h其ROS水平显著升高(11.30±1.67vs 66.70±6.53,P0.05),在加入不同钙离子拮抗剂后ROS水平明显下降(P0.05);与LDL处理组比较,1mmol/L TFAS+LDL处理组MDA水平也显著升高(0.134±0.027vs 0.155±0.025,P0.05)。结论:反式脂肪酸能促进HNP-1的表达,提高钙离子依赖的自由基水平,从而提高ECV304细胞氧化LDL的能力。     

反义寡核苷酸抑制缺氧/再给氧时内皮细胞细胞间粘附分子-1的表达

目的:探讨反义寡核苷酸(AS-ODN)对缺氧/再给氧(H/R)时内皮细胞细胞间粘附分子-1(ICAM-1)表达的影响。方法:流式细胞仪测定肾小球血管内皮细胞在缺氧、再给氧及加入AS-ODN后ICAM-1表达的阳性百分率。结果:缺氧10h,肾小球血管内皮细胞ICAM-1的表达与对照组无显著性差异,再给氧6hICAM-1的表达明显高于正常,加入AS-ODN后,ICAM-1阳性细胞的百分率下降40.6%。结论:AS-ODN可以降低H/R时内皮细胞ICAM-1的表达。     

沉默PARP-1对乳腺癌细胞MCF-7顺铂耐药的影响

目的:探究沉默多聚腺苷二磷酸核糖聚合酶1(PARP-1)对乳腺癌细胞MCF-7顺铂耐药的影响及可能的作用机制。方法:Western blot及real-time PCR实验检测乳腺癌细胞株MCF-7及相应耐药细胞株MCF-7/DDP中PARP-1的表达情况。采用转染PARP-1小干扰RNA(siRNA)的方法沉默乳腺癌耐药细胞株MCF-7/DDP中PARP-1的表达,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测PARP-1、Bcl-2、Bax、cleaved caspase-3、caspase-3、细胞色素C(Cyto-C)、细胞外调节蛋白激酶(ERK)和磷酸化ERK(p-ERK)的蛋白水平。结果:耐药细胞株MCF-7/DDP中PARP-1的mRNA及蛋白表达水平均明显高于亲本细胞株(P0.05);并且在亲本细胞株MCF-7中加入顺铂刺激后,PARP-1的蛋白表达水平明显升高(P0.05)。沉默PARP-1可诱导乳腺癌耐药细胞株MCF-7/DDP的凋亡,增强细胞对顺铂的敏感性,还可下调Bcl-2/Bax和p-ERK的蛋白水平,同时上调cleaved caspase-3及Cyto-C的蛋白水平。而应用ERK特异性抑制剂U0126后,PARP-1沉默组的细胞活力进一步降低。结论:沉默乳腺癌耐药细胞株MCF-7/DDP中PARP-1的表达可恢复细胞对顺铂的敏感性,促进细胞经线粒体途径的凋亡,其机制可能与其抑制ERK的磷酸化,进而阻断该信号通路有关。     

葡萄糖胺抑制LL-37诱导的人脐带血管内皮细胞活化

目的探讨葡萄糖胺对LL-37诱导的人脐带血管内皮细胞(HUVEC)细胞间黏附分子-1(intercellular adhesion molecule,ICAM-1)和单核细胞趋化因子(monocyte chemoattractant factor,MCP-1)表达的影响。方法实时逆转录聚合酶链式反应(Rea ltime RT-PCR)和免疫印迹法(Western blot)、酶联免疫吸附测定(enzyme linked immunosorbent assay)分别测定ICAM-1和MCP-1的表达;Western blot法测定HUVEC蛋白的O-连接N-乙酰葡萄糖胺(O-GlcNAc)修饰及细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)的磷酸化程度。结果葡萄糖胺抑制LL-37诱导的ICAM-1和MCP-1在HUVEC的表达;葡萄糖胺诱导内皮细胞蛋白的O-GlcNAc修饰;葡萄糖胺抑制LL-37诱导的ERK磷酸化。结论葡萄糖胺通过抑制ERK的活化抑制LL-37诱导的ICAM-1和MCP-1的表达。     

HIV-1 Nef stabilizes AP-1 on membranes without inducing ARF1-independent de novo attachment

HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins.     

HIV-1 Nef binding protein expressed on the surface of murine blood cells

The Nef protein of HIV-1 binds to and induces apoptotic cytolysis of a broad spectrum of uninfected blood cells of humans and mice independently of CD95 (Fas). A 24-kDa glycoprotein responsible for Nef binding and the Nef-induced apoptosis has been identified on the surface of human CD4⁺ T cells. Using mouse monoclonal antibodies (mAbs) against the human Nef-binding protein and flow cytometry, we analyzed the expression of a corresponding protein on murine cells. The mAbs were shown to bind to the surface of various murine cell lines including T and B lymphocytes and macrophages, in a fashion similar to the binding by soluble Nef protein. The mAbs competed with the Nef protein in binding to the cell surfaces. Immunoprecipitation of cell membranes revealed a 25-kDa protein recognized by the mAbs. Treatment of the soluble Nef protein with anti-Nef (C terminus) mAb, but not anti-Nef (N terminus) mAb, deprived the Nef of the cell binding activity, indicating that binding site is located in the C-terminal domain. Cross-linking of the cell-bound mAbs with secondary antibodies induced apoptotic cytolysis, which occurred independently of CD95 (Fas). On the other hand, neither the mAbs nor the soluble Nef protein reacted with primary lymphocytes in a resting stage obtained from lymph nodes, thymus and spleen of 5-week-old mice. However, some of the cells, predominantly comprising CD4⁺ T cells, became positive for the both reactions after mitogenic stimulation with phytohemagglutinin, concanavalin A or lipopolysaccharide of Escherichia coli. These results suggest that the 25-kDa protein on murine cell surfaces corresponds to the human Nef binding protein and is responsible for the Nef-induced apoptosis, and that its expression on the cell surface depends on cellular activation. Received: 23 December 1997     

In vivo mutational analysis of the N-terminal region of HIV-1 Nef reveals critical motifs for the development of an AIDS-like disease in CD4C/HIV transgenic mice

HIV-1 Nef is a critical determinant of pathogenicity in humans and transgenic (Tg) mice. To gain a better understanding of the molecular mechanisms by which Nef induces an AIDS-like disease in Tg mice, a mutational analysis of the N-terminal domain, involved in anchoring Nef to the plasma membrane, was carried out. The pathogenic effects of these Nef mutant alleles were evaluated in Tg mice by FACS analysis and by histopathological assessment. Mutation of the myristoylation site (G2A) completely abrogated the development of the AIDS-like organ disease in Tg mice, although partial downregulation of the CD4 cell surface protein and depletion of peripheral CD4+ T-cells, but not of CD4(+)CD8+ thymocytes, still occurred. Despite that, the peripheral CD4+ T cells expressing Nef(G2A) show normal spontaneous proliferation in vivo or after stimulation in vitro, including in an allogenic mixed leukocyte reaction (MLR). Three other internal deletion mutants of Nef, spanning amino acids 8-17 (Nef(Delta8-17)), 25-35 (Nef(Delta25-35)), and 57-66 (Nef(Delta57-66)), were also studied. Nef(Delta8-17) retained full pathogenic potential, although Nef(Delta25-35) and Nef(Delta57-66) Tg mice were free of organ disease. However, Nef(Delta25-35) Tg mice exhibited disorganization of thymic architecture and a partial depletion of peripheral CD4+ T cells. These data indicate that myristoylation and other regions at the N-terminus of Nef (aa 25-35 and 57-66) are involved in mediating severe T-cell phenotypes and organ disease, although residues 8-17 are dispensable for these Nef functions. In addition, these results indicate that at least some of the CD4+ T-cell phenotypes can develop independently of the other AIDS-like organ phenotypes. This apparent segregation of different Nef-mediated phenotypes suggests distinct mechanisms of Nef action in different populations of target cells, and may be relevant to human AIDS.     

HIV-1 Nef in Macrophage-Mediated Disease Pathogenesis

Combined anti-retroviral therapy (cART) has significantly reduced the number of AIDS-associated illnesses and changed the course of HIV-1 disease in developed countries. Despite the ability of cART to maintain high CD4+ T-cell counts, a number of macrophage-mediated diseases can still occur in HIV-infected subjects. These diseases include lymphoma, metabolic diseases, and HIV-associated neurological disorders. Within macrophages, the HIV-1 regulatory protein “Nef” can modulate surface receptors, interact with signaling pathways, and promote specific environments that contribute to each of these pathologies. Moreover, genetic variation in Nef may also guide the macrophage response. Herein, we review findings relating to the Nef–macrophage interaction and how this relationship contributes to disease pathogenesis.     

Consequences of HLA-associated mutations in HIV-1 subtype C Nef on HLA-I downregulation ability

Identification of CD8+ T lymphocyte (CTL) escape mutations that compromise the pathogenic functions of the Nef protein may be relevant for an HIV-1 attenuation-based vaccine. Previously, HLA-associated mutations 102H, 105R, 108D, and 199Y were individually statistically associated with decreased Nef-mediated HLA-I downregulation ability in a cohort of 298 HIV-1 subtype C infected individuals. In the present study, these mutations were introduced by site-directed mutagenesis into different patient-derived Nef sequence backgrounds of high similarity to the consensus C Nef sequence, and their ability to downregulate HLA-I was measured by flow cytometry in a CEM-derived T cell line. A substantial negative effect of 199Y on HLA-I downregulation and Nef expression was observed, while 102H and 105R displayed negative effects on HLA-I downregulation ability and Nef expression to a lesser extent. The total magnitude of CTL responses in individuals harboring the 199Y mutation was lower than those without the mutation, although this was not statistically significant. Overall, a modest positive relationship between Nef-mediated HLA-I downregulation ability and total magnitude of CTL responses was observed, suggesting that there is a higher requirement for HLA-I downregulation with increased CTL pressure. These results highlight a region of Nef that could be targeted by vaccine-induced CTL to reduce HLA-I downregulation and maximize CTL efficacy.     

Human immunodeficiency virus type 1 Nef protein mediates neural cell death: a neurotoxic role for IP-10

HIV-1 Nef is expressed in astrocytes, but a contribution to neuropathogenesis and the development of HIV-associated dementia (HAD) remains uncertain. To determine the neuropathogenic actions of the HIV-1 Nef protein, the brain-derived (YU-2) and blood-derived (NL4-3) Nef proteins were expressed in neural cells using an alphavirus vector, which resulted in astrocyte death (P < 0.001). Supernatants from Nef-expressing astrocytes also caused neuronal death, suggesting the release of neurotoxic molecules by astrocytes. Analysis of pro-inflammatory gene induction in astrocytes expressing Nef revealed increased IP-10 mRNA expression (4000-fold) that was Nef sequence dependent. Recombinant IP-10 caused selective cell death in neurons (P < 0.001) but not astrocytes, and the cytotoxicity of supernatant from astrocytes expressing Nef YU-2 was blocked by an antibody directed against the chemokine receptor CXCR3 (P < 0.001). SCID/NOD mice implanted with a Nef YU-2-expressing vector displayed abnormal motor behavior (P < 0.05), neuroinflammation, and neuronal loss relative to controls. Analysis of mRNA levels in brains from patients with HAD also revealed increased expression of IP-10 (P < 0.05), which was confirmed by immunoreactivity detected principally in astrocytes. Phylogenetic and protein structure analyses of Nef sequences derived from HIV/AIDS patients with and without HAD suggested viral evolution toward a neurotropic Nef protein. These results indicate that HIV-1 Nef contributes to neuropathogenesis by directly causing astrocyte death together with indirect neuronal death through the cytotoxic actions of IP-10 on neurons. Furthermore, Nef molecular diversity was evident in brain tissue among patients with neurological disease and which may influence IP-10 production by astrocytes.     

Disease progression due to dual infection in an HLA-B57-positive asymptomatic long-term nonprogressor infected with a nef-defective HIV-1 strain

We describe the case of an HLA-B57-positive long-term nonprogressor in whom we previously showed that PBMCs accumulated HIV-1 subtype B proviruses defective in the env gene. After more than 10 years of control of infection, plasma viremia increased progressively, with a concomitant loss of CD4⁺ T cells. By phylogenetic analyses of env, nef, vif, and gag sequences obtained at different time points, we suggest here that this patient was initially infected by a putatively attenuated nef-defective variant and that loss of control was due to superinfection with a fully competent virus belonging to the same clade B. At the time of superinfection, its plasma was unable to efficiently neutralize the superinfecting virus and moderate Gag-specific CD8⁺ T-cell responses were observed. This suggests the limited capacity of even a long-lasting natural infection with a nef-deficient HIV-1 strain to elicit immune responses able to prevent and control superinfection with a virus of the same clade.