Stat5b/Survivin信号转导通路调控结肠癌细胞凋亡的机制

目的: 探讨Stat5b/Survivin信号转导通路调控结肠癌细胞凋亡的作用机制。方法: 用阳离子脂质体介导Stat5反义寡核苷酸转染人结肠癌HT29细胞，MTT法检测细胞增殖状态；流式细胞术检测细胞周期与凋亡；EMSA检测Stat5活性；Western blotting检测Stat5、p-Stat5、cyclin D1、Survivin与Bcl-2凋亡家族成员Bcl-2和Bcl-x_L的表达。结果: 转染Stat5反义寡核苷酸后HT29细胞增殖受抑制，凋亡细胞增多，Stat5、p-Stat5与Survivin表达下降，Bcl-2与Bcl-x_L变化不明显。结论: 阻断Stat5通路可以抑制靶基因Survivin表达并诱导结肠癌细胞凋亡。     

酪氨酸磷酸化抑制剂AG490抑制Jurkat T细胞增殖

目的:通过AG490对Jurkat T细胞活化、增殖、周期、凋亡及ICBP90蛋白表达的影响,探讨阻断JAK/STAT信号通路以抑制Jurkat T细胞生长的可能性及其初步机制。方法:以Jurkat T细胞为模型,应用双荧光抗体标记结合流式细胞仪检测AG490对Jurkat T细胞表面分子CD69和CD25表达的影响;利用噻唑蓝(MTT)比色法观察AG490对Jurkat T细胞增殖的影响;采用碘化丙锭(PI)染色检测AG490对Jur-kat T细胞周期的影响;应用Annexin V-FITC和PI双染色检测AG490对Jurkat T细胞凋亡的影响;Western blot检测AG490对Jurkat T细胞中ICBP90蛋白表达的影响以确定其与AG490抑制Jurkat T细胞增殖的关系。结果:随着AG490浓度从1 mmol/L增至30 mmol/L,细胞停滞于G0/G1期,阻止其进入S期和G2/M期,导致Jurkat T细胞ICBP90蛋白的表达显著降低;AG490对细胞的抑制作用于24 h最为明显,抑制率可达27.37%,呈剂量依赖关系;AG490不能明显抑制Jurkat T细胞的活化或促进其凋亡。结论:AG490能明显抑制Jurkat T细胞的生长,其抑制作用可能通过下调Jurkat T细胞ICBP90蛋白的表达与细胞周期阻滞有关,而不是通过促进细胞凋亡而实现。     

黄连素对T淋巴细胞活化和增殖的抑制作用

目的:研究黄连素(Ber)对T细胞体外活化和增殖的影响及作用机制。方法:正常人外周血全血培养,以植物血凝素(PHA)或佛波醇酯(PDB)加离子霉素(Ion)刺激活化淋巴细胞,双荧光染色及溶血获取有核细胞后,以流式细胞仪分析T细胞表达活化抗原CD69和CD25的水平,并以碘化丙锭染色分析细胞周期分布,7-AAD活染分析细胞死亡率。结果:浓度为100μmol/L和50μmol/L的Ber对PDB+Ion或PHA激活T细胞表达CD69有明显抑制,而25μmol/L的Ber抑制效应无显著性;随时间延长,对CD69表达的抑制程度下降;对于CD25表达,上述3个浓度的Ber抑制作用均有显著性,且呈剂量依赖性。同时,这3个浓度的Ber均能明显阻止淋巴细胞进入S期和G₂/M,对细胞周期的抑制作用没有时相特异性。活染分析显示Ber对淋巴细胞无明显细胞毒性。结论:Ber通过干扰早期活化信号转导通路而抑制T细胞活化和增殖,发挥其免疫抑制作用。     

OX40信号上调Survivin表达对CD4+T淋巴细胞增殖存活的影响

目的　探讨OX4 0信号抑制活化后CD4 T细胞凋亡,维持细胞存活及促增殖作用是否与Survivin相关。方法　分离培养小鼠脾脏CD4 T淋巴细胞,以去除T细胞的脾细胞作为抗原呈递细胞,交联CD3单克隆抗体活化T细胞。用免疫印迹法比较不同时相点,加入OX4 0抗体及转染Survivin负显性突变体基因后,CD4 T细胞中Survivin蛋白表达差异。流式细胞仪检测AnnexinV染色后细胞凋亡数量。〔3H〕TdR掺入法测细胞增殖。结果　CD4 T细胞活化后2～8d ,OX4 0抗体刺激可明显增强Survivin表达,并减少2 5 %细胞凋亡;Survivin突变体降低Survivin蛋白表达后使凋亡增加2 2 %。结论　OX4 0信号能够通过上调Survivin表达来抑制CD4 T细胞凋亡,从而维持细胞存活和增殖。     

VEGF上调不同类型细胞Survivin的表达

目的 探讨血管内皮生长因子（VEGF）对结肠癌细胞（LOVO、HCT116）、人脐静脉血管内皮细胞（HUVECs）Survivin表达的影响和存在的差异。方法 VEGF刺激体外培养的LOVO、HCT116、HUVECs并检测细胞周期和凋亡的变化,用RT－PCR测定mRNA表达,Western blot测定蛋白表达,用免疫细胞化学测定Survivin蛋白表达和部位。结果 VEGF可促进肿瘤细胞增殖,在结肠癌细胞中Survivin表达水平较高,在VEGF诱导后表达上调（P<0.05）,主要分布在细胞质;在HUVECs中,Survivin表达不明显,VEGF诱导后表达明显上调（P<0.05）,在细胞质和细胞核中均有较高表达。结论 VEGF上调3种细胞Survivin mRNA和蛋白表达,但在肿瘤和非肿瘤细胞Survivin上调的程度和分布存在差异。     

人巨细胞病毒感染对宿主细胞周期蛋白表达影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0/G1期的人胚肺成纤维细胞（HEL）,分别于感染后0 h、3 h、6 h、12 h、24 h、48 h、72 h、96 h终止培养。用免疫蛋白印迹法（Western Blot）检测CyclinE、CyclinA、CyclinD1蛋白的表达。结果HCMV感染接触抑制细胞12 h时CyclinE蛋白被诱导,感染后24 h出现CyclinE峰值;HCMV不能诱导CyclinA蛋白表达;CyclinD1没有被诱导,且在感染后24 h开始下降。结论HCMV感染同步化于G0/G1期的细胞后,诱导CyclinE蛋白明显升高,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,将细胞周期阻止于晚G1期。     

阻断4-1BB/4-1BBL通路在系统性红斑狼疮患者T淋巴细胞活化中的作用

目的 探讨协同刺激分子4-1BB/4-1BBL在系统性红斑狼疮(systemic lupus erythematosus,SLE)T淋巴细胞活化中的作用机制.方法 应用RT-PCR和Western blot方法检测体外培养20例SLE患者和20例正常对照者T淋巴细胞活化前后及应用抗4-1BB单抗阻断后p38 MAPK和NF-kB表达的变化.结果 SLE患者T淋巴细胞NF-kB mRNA和p38 MAPK mRNA表达及其蛋白水平明显高于正常对照组(P均＜0.01),活化后的表达进一步升高(P均＜0.01).阻断4-1BB/4-1BBL通路后SLE患者T淋巴细胞p38 MAPK mRNA及蛋白水平的表达均明显下降(P均＜0.01),但是NF-kB mRNA及蛋白水平的表达无明显变化(P＞0.05).结论 协同刺激分子4-1BB可能通过p38MAPK信号转导通路促使SLE患者T淋巴细胞的活化与增殖.     

食管癌ECA109细胞中Survivin shRNA干扰对核因子κB表达的影响及调控机制

目的探讨Survivin作为基因表达调控因子,对核因子κB(NF-κB)基因的转录及蛋白水平活性的影响及与NF-κB的调控关系。方法采用Survivin短发夹RNA(shRNA)干扰技术,脂质体转染食管癌ECA109细胞,检测Survivin沉默效果,选择最佳转染浓度;半定量RT-PCR检测Survivin shRNA干扰后食管癌ECA109细胞中NF-κB及其通路的上游调控因子核因子κB抑制蛋白α(IKKα)、IKKβ表达变化;Western blotting方法检测NF-κB蛋白的表达变化;流式细胞术检测干扰之后食管癌ECA109细胞增殖、凋亡指数的变化。结果 Survivin shRNA干扰食管癌ECA109细胞后,Survivin mRNA和蛋白表达明显下调;NF-κB mRNA表达变化无明显差异,但其蛋白磷酸化水平降低,NF-κB上游因子IKKα、IKKβmRNA表达下调;食管癌ECA109细胞凋亡明显增加,G2期细胞比例增加,S期细胞减少,细胞周期受阻。结论 Survivin通过在转录水平影响IKKα、IKKβmRNA表达,调控NF-κB蛋白活化水平,提示Survivin在肿瘤信号调控网络中可作为一个新的基因表达调控因子。     

YAP1调控间质成纤维细胞活化的研究进展

间质成纤维细胞位于实质细胞周围,参与细胞外基质(extracellular matrix,ECM)分泌.该细胞通常处于静止状态,但在炎症和肿瘤等疾病发生时异常活化.成纤维细胞活化受多种基因调控,这些基因通过促进细胞增殖、促进胶原蛋白和纤维蛋白等ECM分泌,导致局部组织过度纤维化,促进疾病进展.Yes相关蛋白1(Yes-associated protein 1,YAP1)是一种转录因子共刺激分子,同时也是多种信号通路的效应分子.YAP1在活化状态成纤维细胞内表达水平较高.且该蛋白通过调控包含生长因子在内的活化相关的基因表达,可促进成纤维细胞活化.因此,YAP1可能是成纤维细胞活化的关键调控因子,下调YAP1表达有望成为纤维化疾病的潜在治疗方式.     

AG490阻断肝癌细胞Stat3信号通路逆转免疫抑制的实验研究

目的：检测Stat3 mRNA在肿瘤细胞中的表达，研究JAK酶抑制剂AG490阻断Stat3信号通路对免疫抑制因子基因表达的影响，探讨应用AG490阻断肿瘤细胞Stat3信号通路逆转免疫抑制的可行性。方法：RT-PCR法检测Stat3 mRNA表达；动态检测AG490作用前后肝癌细胞Stat3 mRNA表达以及VEGF、TGF-β和IL-10等免疫抑制因子基因的表达情况。结果：在所检测的人肿瘤细胞中5种有Stat3 mRNA的表达；AG490阻断肝癌细胞Stat3通路后，免疫抑制因子VEGF、TGF-β和IL-10等的mRNA表达减弱甚至消失。结论：AG490可通过阻断Stat3信号转导通路逆转免疫抑制，发挥抗肿瘤效应。     

IL-16 inhibits IL-5 production by antigen-stimulated T cells in atopic subjects

BACKGROUND: We have previously shown increased expression of the CD4+ cell chemoattractant IL-16 at sites of airway allergic inflammation. Little is known about the significance of IL-16 in allergic inflammation and its role in allergen-driven T-cell cytokine responses. Because IL-16 interacts specifically with CD4+ T cells, we hypothesized that IL-16 released at sites of inflammation may modulate the pattern of cytokines produced by CD4+ T cells. OBJECTIVE: We investigated the effects of exogenous rhIL-16 on cytokine production of PBMCs from atopic and nonatopic subjects in response to antigen and PHA. METHODS: Primary cultures of freshly isolated PBMCs from ragweed-sensitive atopic subjects and nonatopic subjects were stimulated with ragweed or PHA in the presence or absence of rhIL-16. Supernatant levels of IL-4, IL-5, and IFN-gamma were determined by means of ELISA at different time points between 2 and 6 days. Effects of IL-16 on antigen-induced cellular proliferative responses were determined. RESULTS: No IL-4 protein was detected after antigen stimulation of PBMCs from atopic subjects, whereas significant levels of IL-5 were measured on day 6 (median, 534.9 pg/mL). IL-5 secretion was abolished in PBMC cultures depleted of CD4+ cells. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly reduced the amount of IL-5 released (median, 99.8 pg/mL; P <.001). Detectable levels of IFN-gamma (median, 53.3 pg/mL) were identified after antigen stimulation. The addition of rhIL-16 in antigen-stimulated PBMC cultures significantly increased IFN-gamma levels (median, 255.6 pg/mL; P <.05). Effects of rhIL-16 appear to be specific for antigen-stimulated PBMCs in atopic subjects because rhIL-16 did not alter IL-5 or IFN-gamma production in response to PHA nor did rhIL-16 alter cytokine production in nonatopic normal subjects. CONCLUSION: These studies suggest that IL-16 can play a role in regulating the production of cytokines seen in allergic states in response to antigen.     

人IL-31的克隆表达及对表皮角化细胞的影响

目的克隆人IL-31基因,构建真核表达载体,研究人IL-31对表皮角化细胞HaCaT的影响及其作用机制。方法PMA、PHA刺激正常人外周血单个核细胞,提取细胞总RNA,采用RT-PCR克隆人IL-31基因,并将其克隆到真核表达载体pcDNA3.1/myc-His（-）A,进行PCR和双酶切鉴定,并进行序列测定。将重组质粒转染CHO细胞,用RT-PCR和Western blot分析rhIL-31在CHO细胞中的表达。用Ni^2＋树脂纯化his融合蛋白,用不同剂量rhIL-31刺激HaCaT细胞,利用Transwell穿孔板检测HaCaT细胞培养上清对外周血单个核细胞的趋化作用,荧光定量PCR检测rhIL-31对HaCaT表达趋化因子的影响,Western blot检测STAT3磷酸化。结果成功获得全长人IL-31基因,测序正确,经双酶切、PCR和序列测定鉴定,真核表达质粒构建正确,可在CHO细胞中表达;该目的蛋白刺激人表皮角化细胞HaCaT,细胞培养上清对外周血单个核细胞有趋化作用,HaCaT细胞表达趋化因子MDC、TARC、I-309;细胞STAT3磷酸化增加。结论IL-31作用于正常人表皮角化细胞,细胞培养上清对外周血单个核细胞有趋化作用,HaCaT细胞趋化因子表达增加,细胞STAT3磷酸化增加,提示IL-31可通过STAT3发挥作用。     

IL-6和AG490对Burkitt淋巴瘤细胞生长的影响

 目的: 观察IL-6和AG490对Raji细胞生长的影响,探讨信号转导及转录激活因子3(STAT3)及survivin在Burkitt淋巴瘤发生发展中的作用,为寻找淋巴瘤生物治疗的新靶标提供实验依据。方法: 培养Raji细胞,分别加入STAT3的激动剂IL-6和抑制剂AG490,用real-time PCR和Western blot检测STAT3、survivin的表达情况及STAT3的磷酸化,MTT法检查细胞活力,流式细胞术检测细胞凋亡和细胞周期变化。结果: 培养细胞中加入IL-6或AG490后,细胞生长受明显影响并呈现药物浓度依赖关系(P<0.05);与相应的对照组比较,IL-6组Raji细胞中STAT3、survivin mRNA的表达明显升高,AG490组Raij细胞中STAT3、survivin的mRNA表达明显降低。不同浓度的IL-6组之间、不同浓度的AG490组之间,STAT3和survivin mRNA的表达亦有显著差异(P<0.05),且2种基因mRNA表达都呈现出药物浓度依赖关系。p-STAT3、STAT3和survivin的蛋白水平在IL-6作用下表达增高,AG490作用下表达降低;细胞凋亡率在IL-6作用下逐渐降低,在AG490作用下逐渐升高,且都呈现出药物浓度依赖关系;经AG490处理后,G₁期Raji细胞明显增加,S期细胞无明显改变,G₁/S比值增加,而在IL-6组中,S期细胞明显降低。结论: IL-6和AG490对Raji细胞生长有明显影响,STAT3及其下游靶基因survivin的表达改变可能是IL-6及AG490影响Raji细胞生长的重要分子机制。     

Induction of IL-5 expression by IL-2 is resistant to the immunosuppressive agents cyclosporin A and rapamycin

T cell cytokine expression may be induced by the cytokine IL-2 or via the TCR complex. The comparative effects of cytokine- and TCR-mediated signalling on the induction of human IL-5 mRNA were examined. Cytokine mRNA expression was analysed by RT-PCR in fresh peripheral blood mononuclear cells (PBMC) from normal individuals and in populations of activated T lymphocytes, derived from phytohaemagglutinin (PHA)- stimulated PBMC. rIL-2 induced IL-5 expression in PBMC, the kinetics of which were similar to the effects of PHA. rIL-4 induced IL-5 mRNA expression in activated T lymphocytes. IL-5 expression induced by either IL-2 or PHA was completely abolished by the protein synthesis inhibitor cycloheximide. rIL-2-induced IL-5 expression was resistant to cyclosporin A (CsA), whereas IL-5 expression elicited by PHA was inhibited by CsA, at doses as low as 10 ng/ml. Rapamycin (RAP) had no effect on rIL-2-stimulated IL-5 expression, but suppressed IL-5 expression induced by PHA. The inhibitory effect of RAP on PHA-induced IL-5 expression was more apparent at 12 and 24 h after stimulation than at earlier times. The resistance of IL-2 receptor (IL-2R) signalling to CsA and RAP indicates that the IL-2R and the TCR are associated with different pathways regulating IL-5 expression.      

Inorganic arsenic alters expression of immune and stress response genes in activated primary human T lymphocytes

Inorganic arsenic, a carcinogenic environmental contaminant, exerts immunosuppressive effects on human T lymphocytes. In particular, interleukin-2 (IL2) secretion and T cell proliferation are reduced when peripheral blood mononuclear cells (PBMC) from individuals chronically exposed to arsenic are stimulated ex vivo with lectins such as phytohemaglutinin (PHA). However, it is not clear whether the metalloid directly acts on T cells or blocks monocyte-dependent accessory signals activated by PHA. We report that in vitro pre-treatment of PBMC with sodium arsenite (NaAs) reduces IL2 secretion and T cell proliferation induced by PHA, but does not prevent expression of monocyte-derived cytokines (IL1, IL6, TNFα) functioning as lymphocyte-activating factors. In addition, we found that NaAs delays induction of IL2 and IL2 receptor α chain (IL2RA) mRNA levels in human primary isolated T cells activated by PHA. Kinetic analysis showed that NaAs pre-treatment first inhibits, but thereafter markedly increases, induction of IL2 and IL2RA mRNA when T cells are stimulated with PHA for 8 h and 72 h, respectively. We conducted whole genome microarray-based analysis of gene expression in primary T cell cultures derived from independent donors. NaAs systematically and significantly up-regulated a set of 35 genes, including several immune and stress genes, such as IL13, granulocyte-macrophage colony stimulating factor, lymphotoxin α and heme oxygenase-1 (HO-1). Up-regulation of HO-1, a stress and immunosuppressive protein, was rapidly detectable, both in T cells and in PBMC treated with NaAs. Inhibition of the immunosuppressive activity of HO-1 in PBMC however failed to prevent NaAs-dependent inhibition of T cell proliferation induced by PHA. Our findings demonstrate that, at least in vitro, inorganic arsenic acts directly on human T cells and impairs their activity, probably independently of HO-1 expression and monocyte-related accessory signals.     

IL-6和AG490对移植裸鼠的弥漫大B细胞淋巴瘤和伯基特淋巴瘤生长的影响

目的:观察STAT3通路对移植裸鼠弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)和伯基特淋巴瘤(Burkitt lymphoma,BL)的作用。方法:建立OCI-LY8细胞(DLBCL)和Raji细胞(BL)裸鼠移植瘤模型,并分别将其分为对照组、IL-6组和AG490组,测量裸鼠体重和肿瘤体积;应用Western blot和免疫组织化学检测移植瘤组织中p-STAT3、survivin及血管内皮生长因子(VEGF)蛋白的水平;real-time PCR检测瘤组织中survivin和VEGF的mRNA表达。结果:2种细胞株总成瘤率为83.3%(25/30),其中OCI-LY8细胞成瘤率为66.7%(10/15),低于Raji细胞成瘤率100%(15/15)(P0.05)。DLBCL和BL荷瘤鼠IL-6组在第9和10天裸鼠体重和总瘤体积较对照组均增加,2种移植瘤的IL-6组中第1天与第10天瘤体积总差值均明显大于对照组(P0.05)。pSTAT3阳性信号定位于细胞核;survivin和VEGF以细胞浆表达为阳性。与对照组相比,DLBCL移植瘤IL-6组survivin和VEGF明显升高(P0.05),p-STAT3无显著变化;AG490组p-STAT3及VEGF明显降低(P0.05),survivin无显著变化;在BL移植瘤IL-6组,p-STAT3和VEGF蛋白与相应对照相比均显著升高(P0.05),在AG490处理组中3种蛋白均明显降低(P0.05)。IL-6组和AG490组中survivin和VEGF的mRNA表达与对照组相比差异无统计学显著性。结论:IL-6和AG490可能通过STAT3通路影响DLBCL和BL的生长,STAT3通路活化可能通过调控VEGF和survivin表达而影响肿瘤的发生发展。     

Human recombinant IL-4 suppresses the induction of human IL-2 induced lymphokine activated killer (LAK) activity.

The effect of recombinant human interleukin 4 (rhIL-4) on the induction in vitro of human lymphokine activated killer cell (LAK) activity was investigated. Peripheral blood mononuclear cells (PBMC) from normal healthy donors were incubated for 4 days with or without recombinant human interleukin-2 (rhIL-2) in the presence or absence of rhIL-4. LAK activity was measured against the NK-resistant colon adenocarcinoma cell line SW742, and NK mediated cytotoxicity was determined using NK sensitive K562 cells. Unlike previous reports using mouse effector cells, rhIL-4 neither induced LAK activity nor augmented the cytotoxic response induced by rhIL-2. In four out of six experiments there was a significant reduction of rhIL-2 induced LAK in the presence of rhIL-4, accompanied by a reduction of Tac antigen expression by rhIL-2 activated cells. Recombinant hIL-4 failed to influence the effector phase of the activated PBMC against SW742 or K562 targets.     

应用组织芯片检测食管鳞状细胞癌中Stat3信号转导通路的表达及意义

目的　探讨Stat3、CyclinD1和Bcl-2三种Stat3通路上的蛋白在食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）发生中的作用及其在早期诊断中的意义。 方法　制作组织芯片,包含有860例ESCC组织、198例不典型增生组织、201例单纯增生组织及467例癌旁正常上皮组织,应用免疫组化方法检测三种蛋白的表达情况。 结果 （1）ESCC中三种蛋白的表达均显著高于癌旁单纯增生和正常上皮组织（P<0.05）。（2） 随着ESCC分化程度由高到低,Stat3和Bcl-2蛋白的表达逐渐增强,而CyclinD1的表达逐渐减弱。（3）三种蛋白在ESCC中的表达与患者的年龄、性别、发生部位、肿瘤大小及大体分型差异无统计学意义（P>0.05）,与癌组织浸润深度及淋巴结转移有关（P <0.05）。（4）ESCC中Stat3与CyclinD1的表达呈负相关（r=-0.699,P=0.000）;与Bcl-2的表达呈正相关（r=0.660,P=0.000）;但是CyclinD1与Bcl-2的表达无相关性。 结论　Stat3、CyclinD1和Bcl-2可能在ESCC的发生发展和浸润转移中有重要的作用,CyclinD1和Bcl-2的表达检测有助于ESCC的早期诊断。