蜱传脑炎病毒对人单核细胞的致病性北大核心CSCD

【目的】确定蜱传脑炎病毒(Tick-born encephalitis virus,TBEV)对人单核细胞的感染性及对其复制增殖的影响。【方法】用蜱传脑炎病毒感染单核细胞THP-1,观察细胞病变情况。取不同时间点的细胞培养上清,测定病毒滴度,并用Real Time RT-PCR方法检测病毒核酸;用流式细胞法检测细胞感染率,以确定TBEV在THP-1细胞中的复制增殖情况;同时进行细胞活力检测,以确定TBEV感染后THP-1细胞的变化。【结果】TBEV病毒感染THP-1细胞后,可进行复制增殖,流式细胞法可检测到细胞内的病毒,感染病毒后的单核细胞活力显著降低。【结论】TBEV可在单核细胞THP-1中复制增殖,并可造成细胞活力的显著降低,提示单核细胞可能在TBEV感染机体并扩散至各组织器官过程中发挥了重要作用。     

表达EGFP报告基因口蹄疫病毒亚基因组复制子的构建

【目的】构建含有EGFP报告基因的口蹄疫病毒(FMDV)亚基因组复制子系统。【方法】利用融合PCR方法,将EGFP报告基因替换O型FMDV全长c DNA克隆中的前导蛋白Lb和结构蛋白P1基因,构建含有EGFP报告基因的FMDV亚基因组复制子FMDV-EGFP。复制子质粒连续转化、测序检验复制子载体的稳定性。Not I线性化的复制子FMDV-EGFP用脂质体介导法转染表达T7 RNA聚合酶的BSR/T7细胞后,不同时间段观察EGFP荧光表达情况。转染的细胞用流式、间接免疫荧光、RT-PCR和Western blot检测该复制子载体的自主复制能力和口蹄疫病毒蛋白的表达情况。【结果】复制子质粒的连续转化及测序表明报告基因可以稳定存在。FMDV-EGFP复制子转染BSR/T7细胞3 h后在荧光显微镜下能够看到绿色荧光,EGFP荧光信号随着转染时间的延长逐渐增加,并且荧光信号可持续6 d以上。转染24 h后的细胞流式分析显示转染的细胞中有6.0%发出荧光,说明构建的复制子载体能够有效表达EGFP蛋白。另外,间接免疫荧光、RT-PCR和Western blot方法也检测到该复制子RNA在BSR/T7细胞中能够进行自主复制,并且能够表达病毒的非结构蛋白。【结论】含有EGFP报告基因的FMDV亚基因组复制子的成功构建为进一步研究病毒复制、翻译机制及筛选抗病毒药物等奠定了坚实的基础。     

HIV-1潜伏感染体外实验模型研究进展

潜伏感染的静息记忆CD4+T细胞是清除HIV-1病毒的一个重要障碍。处于潜伏状态的病毒多以原病毒c DNA的形式整合至宿主基因组中,但是病毒基因表达处于沉默状态,因此潜伏感染的细胞难以受到病毒的致细胞病变效应或机体特异性细胞毒性T细胞的杀伤,也不易受到抗反转录病毒治疗药物的作用。如何减少潜伏感染的细胞储存库是艾滋病治疗中亟需解决的一个问题。体内及体外HIV-1潜伏感染模型有助于深入了解HIV-1潜伏感染的建立、维持或打破机制,评价潜伏感染再激活剂的活性。在此侧重于介绍采用永生化细胞系、原代静息CD4~+T细胞或活化的CD4+T细胞建立的HIV-1潜伏感染体外实验模型。     

HIV-1感染诱导丝氨酸/苏氨酸蛋白激酶Citron kinase上调机制研究

HIV-1感染可以改变宿主细胞的表达谱,上调和病毒转录复制翻译包装所需的宿主蛋白,使宿主变成更加适应病毒复制繁殖的环境。研究表明丝氨酸/苏氨酸蛋白激酶Citron kinase(citK)可以促进HIV-1病毒的包装释放,所以我们在本文中进一步探讨了HIV-1感染对Citron kinase在自然生理状态下的表达是否有调节作用。我们用含有荧光素酶报告基因的HIV-1假病毒感染外周血单个核细胞(PBMC)和HEK293T细胞系,检测Citron kinase表达的上调情况。此外,将Citron kinase的上游启动子克隆入含荧光素酶报告基因的载体上,检测HIV假病毒感染对Citron kinase启动子的影响。结果显示:HIV-1可以显著提高PBMC细胞中Citron kinase的表达量,而Citron kinase为HIV-1复制包装所需。在原代CD4+T细胞中过表达Citron kinase,HIV-1的复制可以提高2倍以上。沉默Citron kinase的表达,HIV-1病毒产生量显著降低。在HEK293T细胞系中,HIV-1假病毒感染可以使Citron kinase的mRNA的水平提高2.5倍,蛋白表达量提高2.7倍。我们通过将Citron kinase的启动子克隆到含有荧光素酶报告系统的载体上,感染HIV-1假病毒,发现荧光素酶的活性增加。这提示着HIV-1感染通过转录水平上调Citron kinase的表达,从而为病毒创造复制繁殖更有利的宿主环境。     

以J-Lat 11.1为HIV-1潜伏感染模型再激活效果检测方法的初步探讨

目的:比较3种检测方法的优缺点,探索全面评价人免疫缺陷病毒1型(HIV-1)潜伏感染再激活剂的检测方法。方法:以HIV-1潜伏细胞株J-Lat 11.1为潜伏感染模型、豆蔻酰佛波醇乙酯(PMA)为潜伏再激活剂,用流式细胞术检测绿色荧光蛋白(GFP)阳性细胞所占比例,酶标仪检测GFP表达强度,活细胞成像系统检测GFP的动态表达情况。结果:流式细胞术检测显示PMA再激活出GFP阳性细胞的比例随作用浓度的增加(0~10 nmol/L)而增加,但当PMA浓度高于10 nmol/L后变化不再明显;酶标仪检测显示PMA处理后24 h内,GFP荧光强度逐渐增高,之后可在高水平保持至48 h;活细胞成像系统则可以动态反映PMA处理后的再激活过程。结论:可采用流式细胞术检测GFP阳性细胞比例对HIV-1潜伏感染再激活剂进行初步筛选,再结合酶标仪或活细胞成像系统动态监测GFP的表达情况,综合评价再激活剂的作用强度和起效时间。     

可用于辅助蛋白功能研究的人呼吸道合胞病毒双顺反子微型基因组的构建及拯救

【目的】构建可表达增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的人呼吸道合胞病毒(human respiratory syncytial virus,RSV)双顺反子微型基因组cDNA克隆及重组质粒,并进行拯救,以探讨并验证RSV的聚合酶蛋白或辅助蛋白在RSV反向遗传学操作中的作用。【方法】利用全基因合成和分子生物学相结合的方法,在获得可分别表达EGFP和无生物活性蛋白的单顺反子微型基因组质粒pUC57-RSV-EGFP和pUC57-RSV-ORF1的基础上,进一步克隆至pBR322B载体,获得编码EGFP及无生物活性蛋白的双顺反子微型基因组质粒,经限制性内切酶和核酸序列分析正确后,与可表达4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,通过荧光显微镜观察EGFP的表达以及RT-qPCR对EGFP mRNA的转录水平进行定量分析。【结果】成功构建了编码EGFP和无生物活性蛋白的RSV双顺反子微型基因组质粒pBR322B-RSVⅡ-EGFP,经与编码4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,发现4种辅助蛋白对EGFP的表达具有不同的功能活性。【结论】以可表达EGFP报告基因的RSV双顺反子微型基因组重组质粒,实现了对4种辅助蛋白的功能验证,其中M2-1蛋白在双顺反子微型基因组拯救过程中具有转录延长的生物学活性。     

蜱传脑炎病毒跨过血脑屏障的体外实验

【目的】构建蜱传脑炎病毒(Tick-borne encephalitis virus,TBEV)跨血脑屏障研究的体外细胞模型,研究2种不同细胞的TBEV培养物在病毒跨过血脑屏障中的主要差异,从而为进一步TBEV跨血脑屏障的分子机制研究奠定基础。【方法】利用人脑微血管内皮细胞(Human brain microvascular endothelial cells,hCMEC/D3)构建体外血脑屏障的细胞模型。用BHK-21细胞中培养的蜱传脑炎病毒感染人脑微血管内皮细胞,检测TBEV在hCMEC/D3中的复制增殖情况;将TBEV加入体外血脑屏障模型的上层微孔中,用实时荧光定量PCR和噬斑测定的方法检测跨过血脑屏障的病毒量;将感染TBEV的人单核细胞加入血脑屏障模型的上层微孔中,观察渗漏进下层孔中的淋巴细胞,并用实时荧光定量PCR和噬斑测定的方法检测跨过血脑屏障的病毒量。利用伊文思蓝标记的白蛋白确定血脑屏障细胞的渗透率变化。【结果】实时荧光定量PCR和病毒滴度测定结果表明,TBEV不能在hCMEC/D3细胞中复制增殖,也不能直接跨过血脑屏障;然而,人单核细胞THP-1感染TBEV后,尽管单核细胞不能直接携带TBEV跨过血脑屏障,但THP-1中产生的病毒却能跨过血脑屏障模型进入下层孔中,并引起血脑屏障渗透率的增高。【结论】单核细胞有助于TBEV跨过血脑屏障。     

含EGFP报告基因的慢病毒载体的构建及有感染能力病毒的制备

目的:构建含EGFP报告基因的慢病毒表达载体,包装成病毒并测定病毒感染效率。方法:以pEGFP-C1为模板扩增EGFP基因,经PstⅠ和XhoⅠ酶切后与载体连接,得到慢病毒表达载体pLenti6/v5-EGFP;将此载体与包装质粒共转染包装细胞制备病毒,测定不同感染复数(MOI)下病毒的感染效率。结果与结论:构建了含EGFP报告基因的慢病毒表达载体,制备了有感染能力的慢病毒。     

膜锚定Gaussia萤光素酶的细胞标记及生物荧光成像

目的:制备表达膜锚定Gaussia萤光素酶(extGluc)报告基因的慢病毒,用于标记细胞。方法:将报告基因extGluc克隆至慢病毒载体pCCsin.PPT.SFFV.IRES.eGFP.Wpre(VeGFP)中,以聚乙烯亚胺(PEI)介导,将慢病毒包装所需4种质粒(pVeGFP-extGLuc、pMDL、pRev、pVSVG),转染293FT细胞,72 h后收集病毒上清进行浓缩,感染293FT细胞,并用流式细胞仪检测病毒滴度,生物荧光成像和化学发光分析extGluc的表达;之后,用收集的慢病毒感染人单核细胞白血病细胞株U937。结果:对经PCR筛选出的阳性克隆所含质粒进行酶切鉴定,表明extGlu报告基因插入载体中;重组慢病毒包装成功且病毒滴度为5×106 TU/mL;用包装的病毒颗粒感染293FT细胞,生物荧光成像和化学发光证实extGluc的膜定位,且酶活性与细胞数目呈线性相关;病毒颗粒能够感染悬浮细胞U937。结论:包装了extGluc标记的重组慢病毒,可用于标记细胞,为体内监测细胞迁移、聚集和变化提供了一种方法。     

HIV-1基因表达的转录调控

高效抗逆转录病毒治疗（HAART）可以有效地抑制人类免疫缺陷病毒Ⅰ型（HIV-1）的复制及血浆病毒载量,延缓发病进程,改善、提高患者的生活质量和存活时间。但是,一旦停止治疗就会导致血浆病毒血症迅速反弹,HIV-1以原病毒的形式在静息记忆CD4⁺T等细胞中的持续存在是清除HIV-1的一个障碍。HIV-1基因转录的激活与阻抑决定了受感染细胞进入产毒性感染或潜伏感染。本文从原病毒整合位置与转录干扰、细胞转录因子与HIV-1启动子相互作用招募RNA聚合酶起始转录、转录的表观遗传调控和反式激活因子Tat及其相关蛋白促进转录延伸等方面探讨了HIV-1原病毒转录调控机制。     

Induction of NF-KB during monocyte differentiation by HIV type 1 infection

The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.     

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.     

New aspects in the synthesis and secretion of lysozyme by cultured human monocyte cell lines

The main purpose of this study was to investigate lysozyme synthesis and secretion in three human monocyte cell lines: U-937, HL-60, and THP-1, using sensitive fluorescence-based assay of lysozyme activity. PMA and hIFN-γ were evaluated for inducing lysozyme activity. Using well-defined cell lines from the cell culture collection, no lysozyme activity could be detected in the cultured U-937 cells either with or without addition of the inducing factors. These data suggested, contrary to previous reports, that U-937 cell line cannot synthesize or secrete active lysozyme. THP-1 and HL-60 cells were proved to produce enzymatically active lysozyme in increasing amounts with the time course. PMA and hIFN-γ had no significant inducing effect on the production or the release of active lysozyme in THP-1 and HL-60 cells. We showed inhibiting effect of PMA and hIFN-γ on the lysozyme activity, particularly in HL-60 cell line.     

Lymphokine and phorbol (PMA) regulation of complement (C2) synthesis using U937

Human monocytes synthesize large amounts of the second complement component (C2) after incubation with a T-lymphocyte product called monocyte complement stimulator (MCS). The human monocyte-like cell line, U937, also synthesizes C2 and can be stimulated to increase this synthesis by lymphokine-rich culture supernates. Additionally, phorbol myristate acetate (PMA), an agent which induces maturational changes in other macrophage-like cell lines, also stimulates C2 synthesis by U937 cells. Lymphokine and PMA stimulation of C2 secretion by U937 are both reversibly inhibitable by cycloheximide. At optimal concentrations for stimulation of C2 synthesis, PMA inhibits [³H]thymidine incorporation by U937 indicating that increased C2 is not due to increased numbers of U937 cells.     

U937 and THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB4, LTC4, and LTD4. Incubation of these cells with recombinant human interferon-gamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB4, LTC4, and LTD4 in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by these cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (1000 units/ml) and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37 degrees C. The supernatants were deproteinated and assayed by RIA for LTB4 and LTC4 and by RP-HPLC for LTB4, LTC4, and LTD4. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, less than 0.3ng/5 X 10(6) cells. Peripheral blood mononuclear phagocytes from normal volunteers, cultured and challenged in vitro at under identical conditions, released 11.3 +/- 2.9 ng LTB4 and 2.0 +/- 1.5 ng LTC4/10(6) viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n = 3) nor by preincubation with PMA for 120 hours (n = 3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

HMG-CoA reductase inhibitors reduce adhesion of human monocytes to endothelial cells.

HMG-CoA reductase inhibitors (statins) are believed to reduce coronary heart disease by mechanisms in addition to their well-known cholesterol-lowering effect. We studied the effect of these drugs on monocyte cell adhesion to endothelium. Pretreatment of monocytic cells (U937, THP-1, human CD14(+) monocytes) with 0.01-10 microM concentrations of atorvastatin, cerivastatin, or simvastatin significantly reduced cell adhesion to endothelium. In contrast, pretreatment of endothelium with statins did not affect adhesion of monocytes. Adhesion of monocytes to vascular cell adhesion molecule-1-coated dishes was reduced by these drugs. Cerivastatin also reduced PMA induction of NF-kappaB. Since monocyte adhesion to endothelium is an early event in atherogenesis, treatment with statins in prevention of coronary heart disease may have additional salutary effects to lowering of plasma LDL cholesterol. Our results indicate that the reduction of monocyte adhesion by HMG-CoA reductase inhibitors may be considered as a class effect.