B7-1与B7-2对调节人IL-2基因的转录因子NF-κB和AP-1的相同作用

为了解B7共刺激对细胞因子,特别是对IL-2 mRNA及转录因子NF-κB出和AP-1的影响,探讨B7介导的IL-2调节的分子机制,在异基因混合淋巴细胞反应(MLR)体系中分别或联合加入抗B7-1、抗B7-2单克隆抗体和CTLA-4 Ig以阻断B7/CD28信号传导,通过竞争性PCR定量检测其对IL-2和IL-4 mRNA的影响,并初步测定IFN-γ mRNA的改变,同时用转染MHCⅡ类分子及联合转染等量B7-1或B7-2的NIH3T3转基因细胞tDR7,tDR7/B7-1和tDR7/B7-2刺激CD28+T细胞,通过DNA-蛋白结合实验观察B7对IL-2转录因子NF-出和AP-1的影响.结果表明:抗B7-2单抗和CTLA-4 Ig可明显抑制B7介导的IL-2和IL-4 mRNA合成,而抗B7-1单抗仅有轻度抑制作用,2种或3种抗体联合应用时抑制作用相加.MLR 1-6小时,单独tDR7即可诱导NF-κB的表达,联合转染B7早期对其结合活力无明显影响,6小时后tDR7诱导作用减弱,B7却可显著延长tDR7的诱导作用至72小时.tDR7早期同样可诱导AP-1的表达,联合转染B7分子在24小时内对其有一定的抑制作用,而在反应后期可延长tDR7对AP-1的上调作用,B7-1与B7-2间作用未见明显不同.结论:B7通过减少IL-2 mRNA降解和影响基因转录而上调IL-2分泌,并可同时影响多种细胞因子分泌;在转录水平B7-1与B7-2作用未见明显不同,提示两者的功能差异可能与细胞表达和时间动力学不同有关.详细了解B7介导的T细胞免疫耐受的分子机制有助于设计更好的临床方案预防移植排斥和GVHD.     

白细胞介素7增强急性白血病细胞B7分子表达和免疫原性的研究

目的 探讨白细胞介素7(IL-7)对急性白血病(AL)细胞B7分子表达和免疫原性的影响。方法 用流式细胞术检测AL原代白血病细胞R7分子的表达。体外用IL-7诱导白血病细胞。分析其对B7分子蛋白表达的影响，进而用RT-PCR检测B7-1和B7-2 mRNA表达。MTT法检测诱导后白血病细胞对异基因外周血单个核细胞(PBMNC)的刺激作用，并用酶联免疫吸附法(ELISA)测定上清液中1干扰素(IFNγ)含量：应用B7-1、B7-2和W6／32单抗阻断实验研究B7分子刺激PBMNC的机制。结果 11例AL患中B7-1弱阳性3例，B7-2弱阳性1例。IL-7能显刺激白血病细胞B7-1和B7-2表达，呈时间依赖性。IL-7作用于HL-60细胞可诱导B7-1和B7-2 mRNA表达。诱导后的白血病细胞显刺激PBMNC增殖并产生IFNγ。B7-1单抗和W6／32单抗可抑制白血病细胞诱导PBMNC增殖和产生IFNγ，而B7-2单抗无抑制作用。结论 原代AL细胞低表达B7-1和B7-2分子。在体外，IL-7通过诱导白血病细胞B7-1分子表达，刺激淋巴细胞增殖，使PBMNC分泌IFNγ增多，显提高了白血病细胞的免疫原性。在共刺激分子诱导抗白血病T细胞免疫反应巾，B7-1作用显强于B7-2。     

抗B7分子抗体诱导T细胞免疫耐受研究

目的：联合应用抗B7分子的单克隆抗体（单抗）体外诱导T细胞免疫耐受模型，并探讨B7分子在T细胞免疫耐受中的作用及其机制。方法：体外联合应用抗B7单抗诱导T细胞免疫耐受，^3H-TdR掺入法检测T细胞增殖，逆转录－聚合酶链反应（RT－PCR）检测T细胞细胞因子mRNA合成。为了排除其它粘附分子的作用，应用联合转染DR7或（和）B7分子基因的CHO细胞系作为人工抗原递呈细胞（APC）介导混合淋巴细胞反应（MLR）。结果：T细胞增殖实验显示，B7－1（CD80）和B7－2（CD86）单抗单独应用可以部分阻MLR,人中CD86单抗的阻断作用更为明显。两种抗体联合应用时，可以明显阻断MLR。RT－PCR显示：应用抗B7单抗联合阻断后24h,IFN-γ及IL－2 mRNA合成明显减少，而IL－4 mRNA合成较前明显增加。人工APC介导的MLR显示，仅有第一信号（DR7）时也可以使T细胞增殖，但需要达到一定的信号强度。给予DR7信号同时给予共刺激信号CD80可以增强T细胞反应，增强效应可以被抗CD80单抗所阻断。结论：B7分子在T细胞免疫反应中具有重要的作用，可以非特异性增强T细胞免疫反应。阻断B7／CD28信号传导通路，可以诱导T细胞免疫耐受形成，其中CD86分子的作用可能更为重要。抗B7单抗诱导的T细胞无反应性后，向Th2方向分化，这可能是阻断B7分子共刺激后T细胞免疫耐受形成的机制之一。     

转染B7-1基因的Raji和Jurkat细胞诱导细胞因子mRNA表达的研究

为探讨B7共刺激分子在T细胞活化和分化中的作用，利用脂质体导的转基因技术将B7基因导入恶性血液病细胞系Raji和Jrkat细胞，用流式细胞术检测转基因前，后B7-1基因的表达，用RT-PCR检测T细胞表面细胞因子IL-2，IL-4和IFN-γmRNA的表达及这3种细胞因子在转基因后4，12，20及48小时的时间动态变化，结果发现，B7^ Raji细胞可诱导T细胞分泌细胞因子IL-2，IL-4和IFN-γ，B7^ Jurkat细胞可诱导IL-2和IFN-γ的分泌，而B7^-Raji细胞及B7^-Jurkat细胞均未诱导细胞因子的分泌，IL-2和IL-4在T细胞活化4小时即可检测到，IFN-γ在T细胞活化12小时可检测到，在20小时出现峰值，结论：B7-1基因的导入可有效地增强肿瘤细胞的免疫原性，激活T细胞，B7-1在T细胞活化和分化中起着更主要的作用。     

siRNA特异抑制树突状细胞B7基因表达的研究

目的探讨体外合成的小于扰RNA(siRNA)对人树突状细胞(DC)共刺激分子B7基因表达的影响。方法设计并合成了3条siPRNA(siRNA1,siRNA2和siRNAc),在脂质体的介导下转染树突状细胞,于转染后24、48、72h收集细胞,用半定量RT-PCR检测B7-1和B7-2mRNA的变化,用蛋白印迹检测B7-1、B7-2蛋白的表达。结果:转染24、48、72h后,DC B7-1 mRNA均有明显减少(P<0.01),抑制率分别为(50.0±3.63)%、(66.8±4.12)%和(76.6±4.87)%;B7-2 mRNA均有显著降低(P<0.01),抑制率分别为(53.0±3.70)%、(60.5±3.92)%和(73.4±4.46)%,而单纯脂质转染和siRNAc转染对B7-1、B7-2 mRNA表达均无影响(P>0.05)。WestemBlot结果显示转染48、72h后,siRNA1对B7-1蛋白表达的抑制率为(67.3±4.80)%和(80.9±5.23)%;siRNA2后对B7-2蛋白表达的抑制率为(60.7±4.15)%和(74.7±4.63)%。结论siRNA可特异抑制树突细胞B7基因的转录和表达,为进一步研究siRNA诱导移植免疫耐受提供了理论和实验基础,为诱导器官移植免疫耐受提供了新思路和途径。     

编码人B7-2胞外区cDNA的克隆、表达及生物活性鉴定

B7-2分子是共刺激信号系统B7/CD28/CTLA-4中的重要成员之一。为了更深入地探讨B7-2分子在调控T细胞增殖，分化及相关信号传导过程中的作用，本研究通过聚合酶链式反应（PCR）扩增编码人B7-2分子胞外区的cDNA，测序后定向插入多个原核表达载体并诱导目的蛋白表达，用Western印迹和MTT鉴定生物活性。结果表明；通过pGEX-4T-2载体实现了在宿主菌BL-21（DE3）-codenplus-RIL中较高水平的表达，蛋白表达量为20%。经Western印迹和MTT鉴定，所表达及初步纯化的B7-2胞外区重组蛋白能与抗人B7-2单抗发生特异性结合；在抗人CD3单抗的协同下，B7-2胞外区重组蛋白能有效地促进外周血单核细胞的增殖。结论：B7-2胞外区重组蛋白的获得为进一步研究B7-2分子结构与功能的关系奠定了基础。     

吡格列酮抑制实验鼠损伤血管的IL-1β和NF-кB表达及内膜增生研究

摘要：目的：观察吡格列酮对非糖尿病血管损伤模型鼠内膜增生反应、血管组织局部核因子kappa B(NF-кB)和外周血单个核细胞白介素-1β（IL-1β）表达的影响。方法：建立实验鼠颈总动脉通气—干燥法损伤模型，观察吡格列酮干预后实验大鼠颈总动脉血管内膜增生情况，并应用原位杂交技术分析NF-кB的表达和用半定量逆转录多聚酶链反应（RT-PCR）的方法检测外周血单个核细胞IL-1βmRNA的表达。结果：两组损伤血管管腔均较对照侧显著狭窄；实验组和安慰组的对照侧血管均几乎未见NF-Kappa B P65mRNA表达，损伤侧血管在手术后1周见NF-Kappa B P65mRNA表达，且实验组与安慰组表达无显著性差异，第2周时两组均较第1周时表达更为显著（P<0.05），且安慰组相对实验组表达增加；吡格列酮干预后，实验组IL-1βmRNA的相对半定量值较安慰剂组差异具有显著性（P<0.05）。结论：1． 通气-干燥法血管损伤后外周血单个核细胞IL-1βmRNA表达及血管局部NF-кB原位表达增加、血管内膜面积增大。2. 外周血单个核细胞IL-1βmRNA和血管壁局部NF-кB表达水平与血管内膜面积存在正相关关系。3. 吡格列酮抑制损伤血管的内膜增生及NF-кB原位表达和外周血单个核细胞IL-1βmRNA的表达，且不依赖其代谢调节作用。     

全身炎症反应综合征-急性肺损伤大鼠肺组织IL-4 mRNA表达及AP-1活性变化的研究

目的 复制脂多糖（LPS）致伤的全身炎症反应综合征（SIRS）-急性肺损伤（ALI）的大鼠模型。观察肺组织白介素-4（IL-4）mRNA表达量和激活蛋白-1（AP-1）活性的变化，探讨SIRS-ALI中抗炎机制及其调控的意义。方法 经Wistar大鼠尾静脉注射递增剂量LPS，复制SIRS-ALI大鼠模型；逆转录PCR法（RT-PCR）检测肺组织IL-4mRNA表达量；凝胶迁移率分析法（EMSAs)检测肺组织AP-1活性。结果 （1）LPS可以介导大鼠发生SIRS-ALI；（2）LPS≥6mg/kg可致SIRS-ALI失控，形成ARDS；（3）LPS可诱导肺组织IL-4mRNA表达量和AP-1活性升高；（4）LPS≥6mg/kg时，肺组织IL-4mRNA表达量和AP-1活性的升高幅度最大。结论 （1）LPS≥6mg/kg是大鼠SIRS-ALI发生失控的临界剂量；（2）SIRS-ALI失控伴有IL-4基因转录水平明显上调，可能与上游调控因子AP-1活性异常增强有关；（3）抗炎机制增强在SIRS-ALI发生，发展过程中发挥了重要作用。     

mB7-1重组真核载体的构建及表达

目的构建mB7-1真核表达载体并检测其在B16细胞中的瞬时表达情况。方法提取小鼠脾细胞mRNA,PCR法获得mB7-1片段,与pcDNA3连接,并进行酶切鉴定及测序。重组质粒经脂质体介导转染鼠B16细胞,检测基因表达情况。结果完成mB7-1-pcDNA3重组载体连接及鉴定,基因测序与Genebank一致,并转染至B16细胞,检测到基因表达。结论成功构建mB7-1-pcDNA3真核表达载体,获得mB7-1表达细胞系,为研究其抗瘤效应奠定了基础。     

白血病细胞B7-1分子表达的研究

Ｂ7 1分子是目前研究较多 ,较重要的共刺激分子 ,它的缺乏或封闭可抑制Ｔ细胞的活化、增殖 ,诱导克隆无能。许多动物实验转染外源Ｂ7 1分子基因给肿瘤细胞后 ,成功地诱导了抗肿瘤免疫反应。大多数肿瘤细胞无Ｂ7 1分子的表达。但是一些Ｂ细胞起源的造血干细胞肿瘤有Ｂ7分子的表达。我们分别用逆转录 聚合酶链反应 (ＲＴ ＰＣＲ)和流式细胞术两种方法检测Ｂ7 1ｍＲＮＡ和ＣＤ80 抗原在白血病细胞中的表达 ,并初步探讨其意义。对象和方法1　研究对象　本院门诊和住院患者 5 8例 ,其中急性髓系白血病 (ＡＭＬ) 19例 ,混合性白血病 1例 ,慢性髓系…     

The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter

Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at -150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.     

细胞因子对白血病细胞B7-1基因表达及诱导外周血T细胞活化的影响

目的：观察γ干扰素（ＩＦＮγ）、白细胞介素（ＩＬ）１０、ＩＬ１２对转染Ｂ７１白血病细胞Ｂ７１基因表达及其诱导健康人外周血单个核细胞（ＰＢＭＣ）产生ＩＦＮγ、ＩＬ２的影响。方法：用逆转录聚合酶链反应（ＲＴＰＣＲ）和酶联免疫吸附反应（ＥＬＩＳＡ）方法检测ＩＦＮγ、ＩＬ２基因表达和蛋白质合成。结果：发现ＩＦＮγ、ＩＬ１２增强ＰＢＭＣ产生ＩＬ２、ＩＦＮγ，ＩＬ１０抑制其产生，同时发现ＩＦＮγ、ＩＬ１２增强转染Ｂ７１白血病细胞Ｂ７１基因表达，ＩＬ１０抑制其表达。结论：细胞因子ＩＦＮγ、ＩＬ１０、ＩＬ１２影响转染Ｂ７１白血病细胞诱导ＰＢＭＣ产生ＩＦＮγ、ＩＬ２，其可能是通过影响Ｂ７１基因表达实现的     

Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone

To investigate the cooperative effect of B7-1 and IL-12 in the induction of antitumor activity, we have developed retroviral vectors encoding human B7-1, murine IL-12, or both B7-1 and IL-12 coordinately. Murine transformed liver cells (BNL) were engineered to stably express B7-1, IL-12, or both by infection with corresponding retroviruses. No tumor was observed in 20, 75, and 95% of mice receiving, respectively, B7-1-, IL-12-, and B7-1/IL-12-modified tumor cells after 250 days of inoculation. In contrast, injection of parental BNL or BNL/Neo cells resulted in lethal tumor progression in all mice. Protection against rechallenge with parental tumor cells was observed only in mice who had rejected BNL/IL-12, but not in animals that rejected BNL/B7-1 or BNL/B7-1-IL-12. Growth of parental tumor cells was significantly delayed by simultaneous injection in a distant site of irradiated tumor cells engineered to express IL-12 or both B7-1 and IL-12 but not B7-1 alone. BNL/B7-1 and BNL/B7-1-IL-12 showed similar efficacy in these experiments. Antitumor immunity induced by B7, with or without IL-12, was found to depend mainly on CD4+ T cells with a minor contribution of a non-T cell mechanism; whereas the effect of IL-12 was dependent on CD8+ T cells and on non-T cell effectors. Immunization of mice with IL-12-modified BNL cells induced secretion of a Thl pattern of cytokines while immunization with cells expressing both IL-12 and B7-1 resulted in inhibition of IFN-gamma production. Immunization with BNL/B7-1-IL-12 cells in the presence of anti-human B7-1 MAb resulted in restoration of IFN-gamma production to the levels found in animals injected with BNL/IL-12 cells. To summarize, in our model coexpression of B7-1 and IL-12 in tumor cells does not result in improved antitumoral activity as compared with expression of IL-12 alone. This may be related to the fact that B7-1 changes the mechanisms of antitumor immunity and inhibits IFN-gamma production induced by IL-12 in vivo.     

Clarithromycin suppresses lipopolysaccharide-induced interleukin-8 production by human monocytes through AP-1 and NF-kappa B transcription factors

Erythromycin and other macrolides are effective for the treatment of chronic inflammatory airway diseases such as diffuse panbronchiolitis (DPB) and chronic sinusitis. The effect of macrolides in DPB is suggested to be anti-inflammatory rather than antibacterial. We investigated the effects of clarithromycin on interleukin-8 (IL-8) production using human peripheral monocytes and the human monocytic leukaemia cell line, THP-1. Bacterial extracts from Escherichia coli, Pseudomonas aeruginosa and Helicobacter pylori, as well as E. coli-derived lipopolysaccharide (LPS), induced IL-8 production. Clarithromycin suppressed this production in a dose-dependent manner in both monocytes and THP-1 cells (49.3-75.0% inhibition at 10 mg/L). A luciferase reporter gene assay with plasmids containing a serially deleted IL-8 promoter fragment showed that both the activator protein-1 (AP-1) and/or the nuclear factor-kappa B (NF-kapp aB) binding sequences were responsible for the LPS and clarithromycin responsiveness of the IL-8 promoter. Consistently, in an electromobility shift assay, LPS increased the specific binding of both AP-1 and NF-kappaB, whereas clarithromycin suppressed it. Moreover, LPS and clarithromycin regulated three other promoters that have either the NF-kappa B or the AP-1 binding sequences: two synthetic (pAP-1-Luc and pNF-kappa B-Luc) and one naturally occurring (ELAM-Luc). Our results indicate that clarithromycin modified inflammation by sup-pressing IL-8 production and that clarithromycin may affect the expression of other genes through AP-1 and NF-kappa B. In addition to treatment of airway diseases, the anti-inflammatory effect of macrolides may be beneficial for the treatment of other inflammatory diseases such as chronic gastritis caused by H. pylori.     

Modulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phosphatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor

Shp-2, a src homology (SH)2-containing phosphotyrosine phosphatase, appears to be involved in cytoplasmic signaling downstream of a variety of cell surface receptors, although the mechanism is unclear. Here, we have determined a role of Shp-2 in the cytokine circuit for inflammatory and immune responses. Production of interleukin (IL)-6 in response to IL-1 alpha or tumor necrosis factor (TNF)-alpha was nearly abolished in homozygous mutant (Shp-2(-/)-) fibroblast cells. The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis. In contrast, IL-1/TNF-stimulated nuclear factor (NF)-kappa B DNA binding activity and inhibitor of kappa B (I kappa B) phosphorylation was dramatically decreased in Shp-2(-/)- cells, while the expression and activity of NF-kappa B-inducing kinase (NIK), Akt, and I kappa B kinase (IKK) were not changed. Reintroduction of a wild-type Shp-2 protein into Shp-2(-/)- cells rescued NF-kappa B activation and IL-6 production in response to IL-1/TNF stimulation. Furthermore, Shp-2 tyrosine phosphatase was detected in complexes with IKK as well as with IL-1 receptor. Thus, this SH2-containing enzyme is an important cytoplasmic factor required for efficient NF-kappa B activation. These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-kappa B pathway in a MAP kinase-independent fashion.