人IL—3mRNA的诱生及调节的研究

用[~(35)S]-h-IL-3寡核苷酸探针对人淋巴细胞质RNA进行斑点印迹杂交表明:静止的GO期淋巴细胞内几乎没IL-3mRNA,但在离体以PHA、PHA与PMA或Ⅱa培养细胞,能明显诱导IL-3mRNA产生,提示IL-3基因表达的诱导可能与PK-c、C-AMP及Ca++等第二信使系统和fos-蛋白的反式调节有关。     

人参三醇型皂甙调整骨髓受抑模型鼠的免疫功能

本文探讨了人参三醇型皂甙(Panaxatriol Ginsenoside,PTGS)对环磷酰胺(Cyclophosphamide,CY)造成的骨髓受抑模型鼠免疫功能的调整效应。给小鼠腹腔注射150 mg/kg CY造成骨髓抑制动物模型。注射CY前3天给小鼠皮下注射PTGS可部分恢复CY造成的骨髓细胞数减少及其细胞活力下降,明显提高受抑小鼠IL-1、IL-3、IL-6样物质的产生水平,促进受抑小鼠脾淋巴细胞对ConA的反应性。     

人IL-2mRNA的特性研究

通过测定人IL-2和IL-2mRNA的诱生动力学,在高峰时相从PHA+TPA刺激的人脾脏单个核细胞内用冷酚法提取了大量富含IL-2mRNA的胞浆RNA。用制备型柱状7M尿素—SDS—PAGE分离胞浆RNA,并结合麦胚无细胞转译体系和IL-2生物活性检测,测定出人IL-2mRNA全长片段约为14S。将转译IL-2(tIL-2)与基因工程IL-2(rIL-2)和部分纯化的天然IL-2(nIL-2)进行分子筛层析,证实三种IL-2洗脱峰基本相同,分子量约为14000道尔顿。tIL-2与IL-2R具良好的结合特性。     

尘螨诱导新生儿脐血单个核细胞ICOS与转录因子表达的研究

为了解尘螨(HDM)抗原对新生儿脐血单个核细胞(CBMC)及成人外周血单个核细胞(PBMC)CD3+ICOS+细胞阳性率、转录因子T-bet、GATA-3、Foxp3 mRNA表达水平以及培养上清中IL-4、IL-10、IFN-γ表达水平的影响。用流式细胞术,检测新生儿CBMC、成人PBMC体外经PHA和/或HDM抗原刺激前、后CD3+ICOS+细胞阳性率;用RT-PCR法检测细胞体外经PHA和/或HDM抗原刺激前、后T-bet、GATA-3以及Foxp3 mRNA表达水平;用ELISA法,检测细胞在体外经PHA和/或HDM刺激前、后培养上清液中IL-4、IL-10、IFN-γ表达水平。结果表明,高剂量HDM抗原显著下调CBMC经PHA刺激后的CD3+ICOS+阳性率(P<0.05),同时也显著上调PHA刺激前其T-bet mRNA表达(P<0.05),而显著下调该类细胞经PHA刺激后的Foxp3 mRNA的表达(P<0.05)。也显著增加其PHA刺激前的IFN-γ分泌(P<0.01),减少其PHA刺激后IL-10分泌(P<0.05)。高剂量HDM抗原对CBMC的作用强于PBMC,可下调CD3+ICOS+阳性率,显著上调PHA刺激前CBMC T-bet mRNA表达,增加PHA刺激前IFN-γ的分泌,减少PHA刺激后IL-10分泌,提示早期接触高剂量HDM抗原对机体免疫功能的影响较强,可促进新生儿Th1样反应,高剂量HDM抗原可能通过作用于下调Foxp3 mRNA的表达而下调CD4+CD25+调节性T细胞的功能。     

干扰素β_2/IL-6是人 T 细胞的辅助刺激因子

作者分离出高纯度的人外周血T 细胞,探讨IL-6对T 细胞增殖的作用。PHA 和IL-6共同刺激,可诱导T 细胞明显增殖,3HTdR 掺入量比单纯PHA 刺激高3—7倍。但比IL-2加PHA 或放射线照射的单核细胞加PHA 刺激所致T 细胞的3HTdR掺入值低3倍。T 细胞增殖与IL-6/PHA 呈     

人外周血淋巴细胞IL-2R_α表达特性及动力学

本文研究了PHA诱导人外周血淋巴细胞(PBl)IL-2Rα表达的动力学,并探讨了TPA、5-氮胞苷、羟基脲、放线菌素D和秋水仙素等对PHA作用的影响。新分离的PBL几乎不表达IL-2Rα。接受PHA刺激12h时,IL-2Rα表达即明显增加,72h达高峰,随后逐渐降下,至第七天接近未刺激水平。TPA、5-氮胞苷可增加PHA的作用,羟基脲和秋水仙素不影响,而放线菌素D可抑制pHA的效应。这表明人PBL IL-2Rα表达在转录水平调节,与DNA合成及细胞有丝分裂关系不大。体外培养的淋巴母细胞再次接触上述试剂时与初次接触呈现相同的反应性。     

外源性IL-23对病毒性心肌炎小鼠Th17细胞增殖的影响

目的:观察小鼠重组IL-23(rIL-23)对病毒性心肌炎(VMC)小鼠Th17细胞增殖的影响。方法:BALB/c小鼠腹腔注射柯萨奇病毒B3(CVB3)建立VMC小鼠模型,1周后磁珠分离脾CD4+T淋巴细胞后进行体外培养,分别加入植物血凝素(PHA)与重组小鼠IL-23(rIL-23)或PHA单独刺激进行体外培养5天。流式细胞术测定培养后Th17细胞占CD4细胞的百分比、RT-PCR测定培养细胞IL-17 mRNA的表达、ELISA法测定培养物上清液中IL-17的水平。结果:与单纯PHA刺激比较,rIL-23刺激后Th17细胞明显增加(1.82%±0.17%比4.70%±1.29%),且培养细胞IL-17 mRNA及上清液中IL-17浓度也增加,两组比较有统计学差异(P均<0.05)。结论:外源性rIL-23可以增加并维持Th17细胞的增殖。     

IL-1和TNF对PHA刺激T细胞增殖反应的协同作用

在PHA(1μg/ml)刺激下,IL-1或TNF均能诱发静止T细胞发生增殖反应,其最适浓度为IL-1(50U/ml)和TNF(200U/ml)。利用其最适浓度,联合作用于PHA刺激T细胞,其增殖反应增强了25％,但仍低于PHA联合10％的Mφm所诱发的增殖反应。单克隆抗体anti-Tac(p55)完全阻断了由PHA联合IL-1所诱发的增殖反应,但对PHA联合TNF所诱发的增殖反应只能部份地抑制。这些结果表明,TNF除了同IL-1一样具有通过IL-2途径来诱导T细胞活化外,还有着其他的途径,提示T细胞活化增殖的第二信号可由多种单核因子提供,而最大的增殖反应的发生,则是多种单核因子和其他因素协同作用的结果。     

人参总皂甙诱导人造血基质细胞表达IL-3的实验研究

目的 研究人参总皂甙(TSPG)对人早期血细胞发生的影响及其机理，进一步探讨人参“补气生血”的现代分子生物学机理。方法 采用造血祖细胞体外培养、造血生长因子(HGF)生物活性检测、免疫细胞化学、核酸分子原位杂交技术，研究TSPG对造血基质细胞表达白细胞介素-3(IL-3)的影响及其机理。结果 TSPG体外作用能明显促进人早期髓系多向性造血祖细胞(CFu-Mix)的集落形成；经TSPG诱导制备的人骨髓基质细胞(BMSG)、脐静脉内皮细胞株(EcV304)、单核细胞株(THP)条件培养液对CFU-Mix的增殖分化有明显促进作用；经TSPG诱导后BMSC、EcV304、THP细胞内IL-3的蛋白及mRNA表达显著提高。结论 TSPG能促进人早期血细胞的增殖分化，其机理可能与TSPG诱导人造血基质细胞表达IL-3有密切关系。     

PHA对CIK细胞增殖和免疫表型影响的研究

细胞因子诱导的杀伤细胞（cytokine induced killers,CIK）是人外周血单个核细胞在IL-1、IL-2、抗CD3单克隆抗体（MAbCD3）和IFN-γ刺激下获得的增殖能力、细胞毒作用强的免疫效应细胞。植物血凝素（phytohemaglutinin,PHA）是T细胞的多克隆活化剂。实验采用PHA先刺激外周血单个核细胞24h．再按CIK细胞的传统培养方法继续培养至15d,观察PHA的加入,对CIK细胞增殖和免疫表型有无影响。     

Perforin gene expression in stimulated human peripheral blood T cells studied by in situ hybridization and northern blotting analysis.

In situ hybridization was used here to monitor the mRNA level of the pore-forming protein perforin in mitogen-stimulated primary peripheral blood human T cells. In situ hybridization was performed using sense and antisense ribonucleotide probes specific for this granule mediator. After IL-2 treatment, an increase in perforin mRNA could be detected by 4 h; they peaked at 12 h, and decreased after 24 h. The perforin mRNA was also induced in T cells treated with a combination of phorbol ester PMA plus lectin or OKT3 mAb. This latter induction followed slower kinetics, peaking at 48 h. For all three mitogens used, even at peak induction times less than 10% of T cells were labeled with perforin probe. Similar patterns of mRNA expression were observed for both unprimed T cells and lectin-primed T blasts. The induction response of mRNA due to IL-2 stimulation is probably mediated by the IL-2 receptor p75 chain since its mRNA was upregulated by IL-2 with a kinetics comparable to that associated with an increase of perforin mRNA. The p55 IL-2 receptor chain increased much more slowly than p75.     

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.     

Quantification of ovine cytokine gene expression by a competitive RT-PCR method

A quantitative competitive RT-PCR method was developed in order to measure IL-1beta, IL-4, IL-12, IFNgamma, TNFalpha and G(3)PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G(3)PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNgamma mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1beta mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.     

采用IL－6cDNA控针检测人IL－6基因表达

采用缺口平移技术对ＩＬ—６ｃＤＮＡ进行了生物素和同位素标记，证明两种标记的ＩＬ－６ｃＤＮＡ（３０ｎｇ／ｍｌ）均可检出低至５ｐｇ的同源ＤＮＡ，而模拟探针（５００μｇ／ｍｌ）对高达１００ｎｇ的同源序列无此反应。应用ＩＬ－６ｃＤＮＡ对富含ＩＬ－６ｍＲＮＡ的标本进行斑点杂交或Ｎｏｒｔｈｅｒｎ杂交，证明人参三醇皂甙促进了淋巴细胞ＩＬ—６基因表达（４倍），人胎脾单个核细胞、人胎脑组织匀浆和人胎胰岛细胞内均含有高浓度ＩＬ—６ｍＲＮＡ。     

Detection and quantitation of interleukin-2 from individual cells

In this report we present the use of cell blotting for the detection of interleukin-2 (IL-2)-producing lymphocytes. This is a rapid and sensitive immunochemical method analogous to Western blotting of proteins. When combined with image analysis one can determine the percentages of IL-2 positive cells as well as quantitate the amount of IL-2 surrounding each cell. When bovine lymph node cells (bLNC) were stimulated with the combination of concanavalin A (ConA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h, 46.4 +/- 0.6% stained positive for IL-2 and, on average, each cell produced 0.92 +/- 0.6 pg of IL-2 in 24 h. Phytohemagglutinin (PHA) and TPA-stimulated human peripheral blood mononuclear cells (hPBMC) produced approximately the same amount. 0.86 +/- 0.4 pg of IL-2 per cell in 24 h; 45.6 +/- 3.6% stained positive for IL-2.