一氧化氮-核因子κB信号通路在支气管哮喘患者T细胞功能表达中的作用

目的　探讨NO 核因子κB(NF κB)信号通路在哮喘患者T细胞细胞因子表达及细胞增殖、凋亡中的调控作用。方法　采用细胞原位杂交、电泳迁移率改变试验(EMSA)、免疫荧光、流式细胞术、双抗夹心ELISA、四甲基偶氮唑蓝微量比色法,观察不同浓度的硝普钠(SNP)及二硫代氨基甲酸吡咯烷(PDTC)对IL 4、IL 5、IFNγ表达及T细胞增殖、凋亡的影响。结果　(1)哮喘患者T细胞IL 4、IL 5mRNA和蛋白表达及细胞增殖均较对照组高,IFNγmRNA和蛋白表达及T细胞凋亡率均较对照组低(P<0 05); (2)低浓度的SNP能上调上述3种细胞因子表达,并使T细胞增殖反应增强、凋亡反应减弱;而中、高浓度的SNP却使上述3种细胞因子表达及细胞增殖率呈剂量依赖性地降低,并使T细胞凋亡率增加; ( 3 )PDTC可抑制10μmol/LSNP对IL 5和IFNγ表达的促进作用,增强1mmol/LSNP对IL 5和IFNγ表达的抑制作用,同时PDTC可减弱10μmol/LSNP对T细胞增殖反应的促进作用,增强1mmol/LSNP对T细胞增殖反应的抑制作用; (4)低浓度的SNP能明显增加NF κB活化细胞百分率及NF κB活性(P<0 05),而中、高浓度的SNP却显著减少NF κB活化细胞百分率及NF κB活性(P<0 05)。结论　哮喘患者T细胞细胞因子表达增多及细胞增殖反应的增强、凋亡反应的减弱与NF κB活化的异常增高有关。N     

蛋白激酶C-核因子κB信号转导通道对人肺动脉平滑肌细胞增殖和血管内皮生长因子表达的影响

目的　探讨蛋白激酶C(PKC) 核因子κB(NF κB)信号转导通道对人肺动脉平滑肌细胞 (HPASMCs)增殖和血管内皮生长因子 (VEGF)表达的影响。方法　体外培养HPASMCs ,用工具药PKC激活剂 12 肉豆蔻酰 13 乙酸佛波酯 (PMA)和NF κB抑制剂二硫代氨基甲酸吡咯烷 (PDTC) ,将HPASMCs分为对照组、PMA组和PMA PDTC组在常氧和缺氧条件下培养。逆转录 聚合酶链反应 (RT PCR)检测VEGFmRNA表达 ,Westernblot法检测VEGF和NF κB的抑制蛋白IκBα蛋白表达 ,免疫细胞化学法检测NF κBp6 5的表达和定位 ,流式细胞术检测细胞周期时相分布。结果　( 1)NF κBp6 5胞核染色阳性率、IκBα蛋白相对表达量及细胞周期G2 /M % :常氧或缺氧PMA组与相应对照组、PMA PDTC组比较差异均有显著性 (P均 <0 0 5 ) ;缺氧PMA组与常氧PMA组比较差异有显著性 (P <0 0 5 )。 ( 2 )VEGFmRNA和蛋白表达 :常氧对照组、PMA组、PMA PDTC组组间差异均无显著性 (P均 >0 0 5 ) ;缺氧PMA组均高于缺氧对照组、缺氧PMA PDTC组、常氧PMA组 ,差异均有显著性 (P均 <0 0 5 )。 ( 3)缺氧PMA组NF κB胞核染色阳性率、VEGF蛋白相对表达量、G2 /M %之间均呈正相关 (r =0 5 87～ 0 710 ,P均 <0 0 5 )。结论　常氧培养HPASMCs存在PKC NF κB信号转导通道 ;     

运动性哮喘患者中嗜酸细胞、T淋巴细胞、白三烯作用的研究

目的探讨嗜酸细胞、T淋巴细胞、白三烯和运动性哮喘(EIA)的关系.方法分别测定13例运动性哮喘患者和19例非运动性哮喘患者运动前、后血清中的嗜酸细胞阳离子蛋白(ECP)浓度和白细胞介素4(IL-4)mRNA和白细胞介素5(IL-5)mRNA值以及外周血CD+25T淋巴细胞占总淋巴细胞的百分比(CD+25%);另外测定22例运动性哮喘患者运动前和运动后2h尿液中白三烯E4(LTE4)浓度,给予白三烯受体拮抗剂扎鲁斯特治疗;并与正常对照组进行对照.结果哮喘患者运动前ECP和CD+25%与一秒钟用力呼气容积(FEV1)呈负相关(r分别为-0.79、-0.61,P均<0.01).运动性哮喘组与非运动性哮喘组之间IL-4mRNA、IL-5mRNA、CD+25%、ECP在运动前、运动后10和60min比较差异无显著性(P>0.05).运动性哮喘组、非运动性哮喘组、对照组之间最大分钟通气量(Emax)分别为(73.6±34.2)L/min、(69.1±22.1)L/min、(59.6±23.5)L/min,组间比较差异无显著性(P均>0.05).运动性哮喘组运动2h后尿液中LTE4浓度为(225.7±97.4)ng/L与运动前(152.9±89.4)ng/L比较,差异有显著性(P<0.01);服用扎鲁斯特后,运动性哮喘运动后反应与用药前比较明显减轻,其中运动后1h内FEV1下降与时间形成的曲线下面积[AUC0～60min(用药前为1.0用药后为0.3)]、运动后FEV1恢复至运动前所需时间(用药前为75min∶用药后为45min)、运动前、后FEV1下降的百分比(用药前为-22.9%∶用药后为-6.9%,P均<0.01).结论过度通气并非EIA形成的主要因素;T淋巴细胞的激活以及主要由Th2细胞分泌的细胞因子IL-4、IL-5,嗜酸细胞分泌的ECP在EIA的发生中未起主导作用;白三烯在运动性哮喘的发生中起到重要作用.     

核因子κB在被动致敏的人气道平滑肌细胞增殖中的信号转导作用

目的探讨核因子κB(NF-κB)是否参与被动致敏的人气道平滑肌细胞(HASMC)增殖及是否是蛋白激酶C(PKC)激活后的下游途径.方法用10%哮喘患者血清被动致敏HASMC,以10%非哮喘者血清为对照,并用吡咯烷二硫氨基甲酸(PDTC)及12-肉豆蔻酰-13-乙酸佛波酯(PMA)干预HASMC,用流式细胞术、MTT法及增殖细胞核抗原(PCNA)免疫荧光技术检测HASMC增殖;NF-κBp65免疫荧光技术及电泳迁移率改变分析(EMSA)检测NF-κB活性.结果 (1)哮喘血清处理的HASMC,S期细胞比例、吸光度值(A值)、PCNA表达阳性率、NF-κBp65阳性率及EMSA灰度值均较对照血清组增加(均P<0.05),PDTC预处理后上述指标均下降 (均P<0.05).(2)PMA+哮喘血清处理HASMC后,S期细胞比例、A值、PCNA表达阳性率、NF-κBp65阳性率及EMSA灰度值分别为(25.52±3.38)%、0.572±0.054、 (81.2±10.2)%、(26.5±5.0)%和71 654±12 293,PDTC预处理后上述指标均下降(均P<0.05).结论 NF-κB参与了哮喘血清被动致敏的HASMC增殖,在其增殖中存在着PKC/NF-κB信号途径.     

转化生长因子-β对人Th17细胞增殖的影响

目的探讨转化生长因子（TGF）-β对人Th17细胞的免疫调节作用。方法应用免疫磁珠分离正常人CD4^＋T细胞并随机分为观察组和对照组,均置于含5μg/ml植物凝集素和5 ng/ml人重组IL-2的IMDM培养基中,在此基础上观察组加入100 IU/ml TGF-β,均于37℃、5%CO2孵箱中培养4 d。采用ELISA法检测两组细胞上清中IL-17蛋白水平,流式细胞术检测CD4^＋T细胞内IL-17比例（Th17细胞数量）。结果观察组和对照组CD4^＋T细胞上清中IL-17蛋白水平分别为（6.11±9.20）、（15.07±12.70）pg/ml,观察组低于对照组（P=0.07）;观察组和对照组Th17细胞数量分别为2.45%±0.62%、2.81%±0.44%,观察组明显低于对照组（P〈0.05）。结论 TGF-β在体外能通过抑制人Th17细胞增殖而调节其免疫作用。     

二硫代氨基甲酸吡咯烷对苦参碱诱导肝癌细胞凋亡的影响

目的 观察二硫代氨基甲酸吡咯烷(PDTC)抑制核因子-κB(NF-κB)活化后对苦参碱诱导肝癌细胞HepG2凋亡的影响.方法 MTT法观察苦参碱(分0.8、1.0、1.5、2.0、2.5 g/L组)及PDTC联合苦参碱对HepG2细胞增殖的抑制作用.将HepG2细胞随机分为细胞对照组、PDTC组(20μmol/L)、苦参碱组(1.5 g/L)和PDTC+苦参碱联合组,流式细胞仪和末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测细胞凋亡;电泳迁移率改变实验检测细胞核内NF-κB的活化水平.结果 PDTC增强了苦参碱对细胞增殖的抑制作用(F=183.92,P＜0.01).苦参碱同时具有诱导HepG2细胞凋亡和NF-κ B活化的作用;PDTC能显著增加苦参碱诱导的HepG2细胞凋亡和抑制苦参碱诱导的HepG2的NF-κB活化,细胞凋亡率由6.11%±0.81%增加至12.95%±0.02%(χ2=9.67,P＜0.05),NF-κB活化的灰度值由38.82±0.17降至32.01±0.69(χ2=10.38,P＜0.05).结论 苦参碱诱导HepG2细胞凋亡的同时激活NF-κB;PDTC可通过抑制NF-κB活化,增强苦参碱诱导HepG2细胞凋亡的作用.     

哮喘患者辅助T细胞活化与白细胞介素5释放

目的 了解过敏状态和哮喘状态下辅助（ＣＤ４＾＋）Ｔ细胞活化及白细胞介素５（ＩＬ－５）释放的原因和作用。方法 对过敏性哮喘组１２例（ＡＡ）、非过敏性哮喘组１０例（ＮＡＡ）、过敏性非哮喘组９例（ＡＮ）及正常对照组１０名（Ｎ）在有无抗原刺激下进行支气管肺泡灌洗液（ＢＡＬＦ）细胞及周围血单个核细胞（ＰＢＭＣ）培养，比较组内和组间ＣＤ４＾＋Ｔ细胞活化（标志物ＣＤ２５＾＋）与ＩＬ－５释放水平。结果 ＰＢＭＣ在     

Increased expression of CD30 and CD57 molecules on CD4(+) T cells from children with atopic asthma: a preliminary report.

T helper type 2 (Th2) cells play an important role in the onset and persistence of allergic airway inflammation. Consequently, many authors have attempted to identify cell surface markers associated with a Th2 phenotype. This work was aimed at correlating CD30 and CD57 expression on CD4(+) T cells with interleukin (IL)-4 production in peripheral blood mononuclear cells (PBMCs) from allergic patients. PBMCs from 17 children with atopic asthma and 12 nonatopic healthy control children were analyzed. The CD28, CD30, CD40L, CD57, CD62L, CD69, IL-4, and IFN-gamma expressions on CD4(+) T cells were determined by double immunofluorescence and flow cytometry in PBMCs ex vivo and after phorbol-12-myristate-13-acetate plus ionomycin (PMA/I) stimulation. An increased percentage of peripheral CD4(+)CD30(+) T cells was observed in asthmatic patients (p < 0.001). In addition, the percentage of CD4(+) T cells expressing IL-4, IFN-gamma, CD30, CD40L, CD57, or CD69 significantly increased (p < 0.01) after PMA/I stimulation, in asthmatic patients. The CD30 expression on CD4(+) T cells from asthmatic patients, after stimulation, correlated with both IL-4 and IFN-gamma production, whereas CD57 expression only correlated with IL-4 production. These data suggest that the expression of CD30 and CD57 cell markers on T cells could reflect circulating effector T cell early activation in the allergic airway disease.     

Increased entry of CD4+ T cells into the Th1 cytokine effector pathway during T-cell division following stimulation in Behcet's disease

OBJECTIVES: To investigate the relationship between the production of Th1/Th2 cytokines and cell kinetics, cell division and proliferation in patients with Beh?et's disease (BD). METHODS: Peripheral venous blood was drawn from patients with BD (n = 24; 10 patients with active and 14 patients with inactive BD) and normal subjects (n = 22). Peripheral blood mononuclear cells were separated immediately and were cultured with concanavalin A (Con A) followed by phorbol 12-myristate 13-acetate and ionomycin (PMA+Ion). Intracellular cytokine production of interferon-gamma (IFN-gamma) (Th1) and IL-4 (Th2) in CD4(+) T cells was determined by flow cytometry. Furthermore, CD4(+) T cells labelled with CFSE [5 (and 6) carboxyfluorescein diacretate, succinimidyl ester] were stimulated and the cells were analysed for entry into the cytokine production effector pathway during cell division in active BD and normal subjects. RESULTS: In active BD, enhanced entry into the Th1 response effector pathway of CD4(+) T cells was observed after stimulation with Con A followed by PMA+Ion. Analysis of CD4(+) T cells at an identical cell division number in response to Con A followed by PMA+Ion revealed that IFN-gamma-producing cells were increased in active BD patients compared with normal subjects. These results suggest that the Th1 response of dividing CD4(+) T cells is predominantly operating in active BD. Dividing CD4(+) T cells stimulated with Con A followed by PMA+Ion showed a phenotype of activated effector memory T cells (CD45RA(low), CD45RO(+), CD69(high)). CONCLUSIONS: Cell kinetics play a crucial role in Th1 cell differentiation and pathophysiology in BD.     

蛋白激酶Cα-胞外调节蛋白激酶1/2与人气道平滑肌细胞中周期蛋白D1及P21cip1 表达的关系

目的 探究蛋白激酶C(PKC)α-胞外调节蛋白激酶(ERK)1/2级联对支气管哮喘(简称哮喘)患者血清被动致敏的人气道平滑肌细胞(HASMC)周期蛋白D1(cyclinD1)、周期蛋白依赖性激酶(CDK)抑制剂P21 cipl的调节作用及细胞增殖的影响.方法 用含10%哮喘患者血清的DMEM培养基被动致敏HASMC后,按随机数字表法分为对照组、12-肉豆蔻酰-13-乙酸佛波酯(PMA)处理组、PMA+PKCα错配寡核苷酸组、PMA+PKCα反义寡核苷酸组以及PMA+U0126组,每组4个样本.用Western blot方法检测各组细胞磷酸化PKCα(p-PKCα)、ERK1/2、磷酸化ERK1/2(p-ERK1/2)、cyclinD1和P21 cipl的蛋白表达水平,用流式细胞术和四甲基偶氮唑盐(MT)法检测HASMC增殖.结果 PMA处理组p-PKCα水平、ERK1/2和p-ERK1/2蛋白水平高于对照组,cyclinD1、P21 cipl表达明显强于对照组(相对于各对照组的A值分别为2.10±0.29、1.67±0.19、2.20±0.27、1.99±0.22和3.11±0.29,均P＜0.05),HASMC的增殖[S期细胞比例为(30.3±2.4)%,A490值为0.80±0.06]高于对照组[S期细胞比例为(13.9±2.6)%,A490值为0.41±0.04],均P＜0.05;PMA+PKCα反义寡核苷酸组中p-PKCα水平低于PMA处理组,ERK1/2和p-ERK1/2表达明显弱于PMA处理组,cyclinD1和P21 cipl 表达也明显低于PMA处理组(相对于各对照组的A值分别为1.23±0.19、1.34±0.18、1.52±0.20、1.45±0.18和1.49±0.18,均P＜0.05),HASMC的增殖显著下降[S期细胞比例为(21.2±2.8)%,A490值为0.51±0.04;q值分别为6.07,12.63;均P＜0.05];同样与PMA处理组相比,PMA+U0126组中p-PKCα水平无明显改变(A值为1.99±0.18,q=0.94,P＞0.05),但ERK1/2、P-ERK1/2的表达,cyclinD1和P21 cipl的表达明显低于PMA处理组A值分别为0.95±0.21、1.15±0.19、1.37±0.15和1.96±0.21,均P＜0.05),HASMC的增殖显著下降[S期细胞比例为(22.0±3.2)%,A 490值为0.49±0.03;q值分别为5.51、13.45,均P＜0.05].结论 ERK1/2是PKCα的下游信号分子,PKCα-ERK1/2级联参与了PMA所诱导的哮喘患者血清被动致敏的人气道平滑细胞cyclinD1、P21 cipl的表达上调及细胞增殖.     

咪喹莫特治疗支气管哮喘作用机制的实验研究

目的 研究新型免疫调节剂咪喹莫特对支气管哮喘(简称哮喘)的治疗作用,探讨其可能的作用机制.方法 (1)40只BALB/c小鼠(每组10只)和48只SD大鼠(每组12只)分为对照组、哮喘组、咪喹莫特组和地塞米松组,建立大鼠和小鼠哮喘模型.用卵清白蛋白激发前吸入0.15%咪喹莫特混悬液5 ml.末次激发后24 h观察肺组织炎症及测定气道反应性;用逆转录-聚合酶链反应(RT-PCR)法测定肺组织中白细胞介素4(IL-4)、γ干扰素(IFN-γ)、嗜酸粒细胞活化趋化因子(eotaxin)、巨噬细胞衍生趋化因子(MDC)、胸腺和活化调节趋化因子(TARC)、T-bet、GATA-3、信息转导转录激活子6(STAT6)mRNA的表达;用酶联免疫吸附试验(ELISA)法测定血清中eotaxin、MDC、TARC及支气管肺泡灌洗液中IL-4和IFN-γ浓度;用Western blot法测定肺组织中T-bet、GATA-3、STAT6蛋白水平;(2)分离和培养致敏大鼠气管旁淋巴结细胞(PBLN),分为空白对照组、阳性对照组、地塞米松组和药物1～3组,测定不同时间点各组细胞上清液中IL-4、IFN-γ蛋白和细胞的mRNA表达;(3)分离和培养致敏小鼠的脾脏T淋巴细胞,用流式细胞仪测定经咪喹莫特刺激后不同时间点细胞内IL-4、IFN-γ水平;(4)培养致敏大鼠CD+4T淋巴细胞,测定经咪喹莫特干预后T-bet、GATA-3 mRNA和蛋白的表达水平.结果 用同等剂量氯化乙酰胆碱(20、40、80、160μg/ml)激发时平均呼气阻力哮喘组分别为(6.26±0.85)、(11.55±3.09)、(28.74±5.94)、(3710.83±197.49)cm H20·ml-1·s-1(1 cm H20=0.098 kPa),咪喹莫特组分别为(1.34±0.16)、(3.47±0.49)、(9.29±1.27)、(25.22±5.44)cm H2O·ml-1·s-1,两组相同剂量比较差异有统计学意义(D值分别为88.98、56.00、45.00、108.00,P均＜0.01);哮喘组大鼠气道壁和肺组织中嗜酸粒细胞(EOS)、淋巴细胞、管壁面积/支气管管腔内周长(WA/Pi)、支气管平滑肌面积/支气管管腔内周长(ASM/Pi)分别为(26.0±1.6)、(45.2±3.2)个/mm2、12.0±1.4、6.7±0.6,咪喹莫特组分别为(12.4±2.9)、(24.2±3.7)个/mm2、9.2±0.6、4.0±0.5,两组比较差异有统计学意义(D和q值分别为193.00、16.92、185.50、7.66,P均＜0.01);哮喘组肺组织中T-bet mRNA和蛋白表达量分别为0.08±0.12、0.18±0.06,咪喹莫特组分别为0.48±0.08、0.48±0.17,两组比较差异有统计学意义(D值分别为120.96、177.98,P均＜0.01);哮喘组肺组织中GATA-3 mRNA和蛋白表达量分别为1.56±0.28、2.25±0.74,咪喹莫特组分别为0.54±0.14、0.50±0.14,两组比较差异有统计学意义(D值分别为166.96、310.97,P均＜0.01);空白对照组24、48 h时PBLN细胞培养液中IL-4、IFN-γ分别为(0±0、0±0、22±5、31±5) pg/ml.随着培养时间的延长,药物2组24、48 h时IL-4、IFN-γ水平分别为(23±5)、(39±11)、(149±31)、(154±28) pg/ml,药物3组24、48 h时IL-4、IFN-γ水平分别为(25±6)、(40±12)、(166±30)、(158±31) pg/ml,与阳性对照组[(43±13)、(56±12)、(100±22)、(112±33) pg/ml)]比较差异有统计学意义(药物2、3组24、48 h时IL-4的D值分别为9.90、8.79、8.80、8.10;药物2、3组24、48 h时IFN-γ的q值分别为4.80、6.40、3.95、4.31,P均＜0.05);哮喘组小鼠BALF中细胞总数及EOS分别为(8.7±1.4)×106 /L、(29.80±7.00)%,咪喹莫特组为(4.1±1.3)×106 /L、(8.90±2.40)%,两组比较差异有统计学意义(q值分别为16.40、7.40,P均＜0.05);哮喘组小鼠血清eotaxin、MDC和TARC水平分别为(593±41)、(170±20)、(221±25) pg/ml,咪喹莫特组分别为(501±76)、(84±13)、(163±35) pg/ml,两组比较差异有统计学意义(q值分别为3.70、9.80、4.50,P均＜0.05);(4)咪喹莫特组小鼠肺组织eotaxin、MDC、TARC mRNA表达分别为0.65±0.17、0.66±0.12、0.66±0.34,哮喘组分别为0.85±0.11、0.96±0.10、0.94±0.28,对照组分别为0.45±0.08、0.39±0.09、0.24±0.08,哮喘组与咪喹莫特组比较差异有统计学意义(q值分别为1.50、8.10、2.40,P均＜0.05);哮喘组与对照组比较差异有统计学意义(q值分别为3.00、15.40、5.90,P均＜0.05).结论 咪喹莫特可能通过提高转录因子T-bet mRNA\蛋白的表达和抑制转录因子GATA-3和STAT6 mRNA和蛋白表达,使失衡的Th1/Th2细胞得以纠正,从而抑制哮喘气道炎症.     

法舒地尔对支气管哮喘小鼠肺组织Rho激酶-1表达及气道炎症的影响

目的 观察Rho激酶-1抑制剂法舒地尔对支气管哮喘(简称哮喘)小鼠肺组织Rho激酶-1表达及气道炎症的影响,探讨Rho激酶-1在哮喘气道炎症中的作用机制.方法 将24只BalB/c小鼠采用随机数字表法分为对照组、哮喘组和干预组,每组8只.哮喘组、干预组小鼠分别给予卵清白蛋白(OVA)致敏和激发.每次雾化前1 h,干预组给予法舒地尔(10 mg/kg)腹腔注射.末次激发后收集BalF,离心后计数细胞总数及嗜酸粒细胞(EOS)数量.ELISA法测定BalF上清液中嗜酸粒细胞趋化因子(Eotaxin)、白细胞介素(IL)-5和IL-13水平.肺组织HE染色.采用逆转录PCR和免疫组织化学测定各组小鼠肺组织中Rho激酶-1 mRNA和蛋白的表达水平.结果 (1)哮喘组BalF中细胞总数及EOS数量分别为(1.45±0.12)× 10~9/L和(0.52 ±0.06)× 10~9/L,明显高于对照组[分别为(0.58±0.06)×10~9/L和(0.01±0.01)×10~9/L](q值分别为25.909和35.002,均P＜0.01)和干预组[分别为(0.89 ±0.09)×10~9/L和(0.20±0.04)×10~9/L](q值分别为16.676和21.537,均P＜0.01).(2)哮喘组Eotaxin、IL-5及IL-13水平分别为(45±8)ng/L、(157 ±23)ng/L和(429±46)ng/L,明显高于对照组[分别为(10 ±3)ng/L、(26±6)ng/L和(126 ±20)ng/L](q值分别为18.246、23.009、25.826,均P＜0.01);干预组分别为(20±5)ng/L、(57 ±14)ng/L和(254±28)ng/L,明显低于哮喘组(q值分别为13.119、17.503、8.449,均P＜0.01).(3)对照组小鼠气道周围无炎症细胞浸润,哮喘组小鼠气道黏膜水肿,气道壁及管周有大量以EOS为主的炎症细胞浸润,干预组气道炎症反应较哮喘组减轻.(4)哮喘组肺组织Rho激酶-1 mRNA和蛋白的表达水平明显高于对照组(q值分别为25.614和8.156,均P＜0.01),干预组Rho激酶-1 mRNA和蛋白表达水平低于哮喘组(q值分别为20.379和4.135,均P＜0.01).(5)Rho激酶-1 mRNA表达量与BalF中EOS数量、Eotaxin、IL-5和IL-13水平呈正相关(r值分别为0.709、0.600、0.613、0.650,均P＜0.01).结论 Rho激酶-1参与过敏原诱导的哮喘小鼠气道炎症的发生,应用法舒地尔抑制其表达和活性可能改善哮喘气道炎症.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 mRNAs are produced by a small fraction of blood mononuclear cells

Northern blot analysis has identified granulocyte macrophage colony stimulating factor (GM-CSF) mRNA in monocytes and both GM-CSF and interleukin-3 (IL-3) mRNA in lymphocytes. However, these results have not addressed whether all cells or a subset of the population is capable of hematopoietic growth factor (HGF) production. To resolve this question, we applied in situ hybridization of radiolabeled antisense RNA probes to centrifuged preparations of total blood mononuclear cells (BMCs) and fractionated lymphocyte subpopulations. Without stimulation, no circulating cells expressed detectable levels of GM-CSF or IL-3 mRNA. On stimulation of BMCs with phorbol myristate acetate (PMA) and phytohemagglutinin or PMA and the calcium ionophore ionomycin, approximately 5% expressed GM-CSF mRNA and approximately 1% IL-3 mRNA. Control sense probes produced no labeled cells. To determine the subsets of lymphocytes capable of GM-CSF and IL-3 expression, BMCs were fractionated by FACS into CD8+ and CD4+ lymphocyte subsets and CD16+ (NK) cells. The unfractionated cells and cell fractions were then stimulated with PMA and ionomycin. Results demonstrated that 3% to 5% of the CD16+, CD8+, and CD4+ lymphocytes produced GM-CSF mRNA. However, the number of IL-3 mRNA-positive cells in the FACS-sorted subsets was greatly reduced (0.02% to 0.05%) as compared with the unseparated cells (1%). Treatment of BMCs with high-dose interleukin-2 (IL-2) for 1 week followed by PMA plus ionomycin resulted in a lymphocyte population in which 50% and 3% of cells expressed GM-CSF and IL-3 mRNA, respectively. Thus, GM-CSF and IL-3 mRNA expression in T cells and NK cells is restricted to a small fraction of cells that can be greatly expanded by IL-2 stimulation. These results suggest a possible physiologic mechanism for increasing HGF production by circulating lymphocytes.     

Toll样受体2介导的Th17细胞活化在HBV感染中的作用

目的探讨乙型肝炎患者外周血单个核细胞(PBMC)中Toll样受体2(TLR2)与Th17细胞的相关性,为阐述HBV感染诱导炎症应答机制提供理论和实验依据。方法选取2012年7月-2013年7月唐都医院感染科门诊和住院的34例乙型肝炎初治患者,其中24例慢性乙型肝炎和10例急性乙型肝炎;另外选取健康对照者10例,分离PBMC,利用HBV C基因型Envelope区肽段(特异性)或佛波酯联合伊屋诺霉素(非特异性)刺激,流式细胞术检测TLR2表达及Th17细胞百分比。进一步用TLR2的激动剂刺激PBMC,检测Th17细胞变化情况。组间比较采用Kruskal-Wallis H检验。结果在非特异性刺激条件下,Th17细胞在慢性乙型肝炎患者体内的百分比(4.08±1.78)%显著高于急性乙型肝炎患者(1.85±1.28)%及健康对照者(2.09±0.53)%(P=0.000 9、0.000 4),而TLR2+及IL-17A+TLR2+的表达在急、慢性乙型肝炎患者与健康人外周血中差异均无统计学意义(P均0.05)。在特异性刺激条件下Th17及TLR2的表达在慢性乙型肝炎患者体内的表达显著高于急性乙型肝炎组[(5.45±1.61)%vs(3.20±1.13)%;(5.19±3.18)%vs(1.88±1.30)%],差异具有统计学意义(P=0.000 6、0.000 6)。加入TLR2激动剂后急、慢性乙型肝炎患者体内Th17细胞的比例均显著升高,但在急性乙型肝炎患者中,刺激前后差异无统计学意义(P0.05)。结论 TLR2可以直接影响Th17细胞的应答,从而促进乙型肝炎中炎症应答反应。     

Effect of nuclear factor kappa B on intercellular adhesion molecule-1 expression and neutrophil infiltration in lung injury induced by intestinal ischemia/reperfusion in rats

AIM:To investigate the role of nuclear factor kappa B(NF-κB) in the pathogenesis of lung injury induced byintestinal ischemia/reperfusion (I/R),and its effect onintercellular adhesion molecule-1 (ICAM-1) expressionand neutrophil infiltration.METHODS:Twenty-four Wistar rats weredivided randomly into control,I/R and pyrrolidinedithiocarbamate (PDTC) treatment groups,n=8 ineach.I/R group and PDTC treatment group receivedsuperior mysenteric artery (SMA) occluding for 1 h andreperfusion for 2 h.PDTC group was administrated withintraperitoneal injection of 2% 100 mg/kg PDTC 1 hbefore surgery.Lung histology and bronchia alveoluslung fluid (BALF) protein were assayed.Serum IL-6,lungmalondialdehyde (MDA) and myeloperoxidase (MPO) aswell as the expression level of NF-κB and ICAM-1 weremeasured.RESULTS:Lung injury induced by intestinal I/R,wascharacterized by edema,hemorrhage and neutrophilinfiltration as well as by the significant rising of BALFprotein.Compared to control group,the levels of serumIL-6 and lung MDA and MPO increased significantly in I/Rgroup (P=0.001).Strong positive expression of NF-κBp65 and ICAM-1 was observed.After the administrationof PDTC,the level of serum IL-6,lung MDA and MPOas well as NF-κB and ICAM-1 decreased significantly(P<0.05) when compared to I/R group. CONCLUSION:The activation of NF-kB plays animportant role in the pathogenesis of lung injury inducedby intestinal I/R through upregulating the neutrophilinfiltration and lung ICAM-1 expression.PDTC as aninhibitor of NF-kB can prevent lung injury induced byintestinal I/R through inhibiting the activity of NF-kB.     

白细胞介素-17在狼疮性肾炎外周血单个核细胞上的表达及意义

目的　探讨白细胞介素 - 17(IL - 17)在狼疮性肾炎患者 (LN)外周血单个核细胞 (PBMC)表达和分泌及其与LN疾病活动性的关系。方法　用酶联免疫吸附法 (ELISA)检测LN活动期患者血浆及PBMC体外自发培养或刺激培养的上清液中IL - 17蛋白水平 ,流式细胞仪免疫荧光分析法检测PBMC表达IL - 17的水平 ;用逆转录 -聚合酶联反应 (RT -PCR)法测定LN活动期患者PBMC体外自发培养和刺激培养后的IL - 17mRNA的表达水平。结果　LN活动期患者血浆检测不出IL - 17(<5ng/L) ;PBMC自发培养和PHA刺激培养下 ,LN活动期、LN静止期和正常对照 ,三组均检测不出IL - 17;而PBMC在CD3mAb +CD2 8mAb和PMA +ionomycin刺激培养下 ,较自发培养和PHA刺激培养 ,上述 3组IL - 17水平均明显升高 (P <0 .0 1) ,LN活动期PBMC表达、分泌IL - 17水平与SLE疾病活动指数 (SLEDAI)呈明显正相关。结论　LN活动期患者PBMC表达、分泌IL -17水平存在明显异常 ,并能反映LN疾病活动程度     

地塞米松对哮喘小鼠CD4+CD25+调节性T细胞及IL-4、IL-10水平的影响

目的 探讨地塞米松对哮喘小鼠CD4+ CD25+调节性T细胞及IL-4、IL-10水平的影响.方法 30只雄性BALB/c小鼠随机分为三组:正常对照组、哮喘组和地塞米松组.利用卵清白蛋白腹腔注射和雾化吸入制备哮喘模型;通过流式细胞仪检测各组小鼠脾脏单个核细胞CD4+ CD25+调节性T细胞占CD4+T细胞的百分比;使用免疫组织化学方法分析各组IL-4在小鼠肺组织中的表达情况;用酶联免疫吸附试验检测各组小鼠血清IL-10的水平.结果 哮喘组脾脏单个核细胞CD4+CD25+调节性T细胞百分比及IL-10的表达水平较正常对照组和地塞米松组降低(P＜0.05),哮喘组IL-4水平较正常对照组和地塞米松组增高(P＜0.05).结论 地塞米松的抗炎作用可能通过上调CD4+ CD25+调节性T细胞、调节性T细胞亚群失衡的途径来实现.     

核因子κB顺式"诱骗"元件对支气管哮喘患者T淋巴细胞功能的干预作用

目的探讨核因子κB(NF-κB)顺式诱骗寡脱氧核苷酸(ODN)对体外支气管哮喘(简称哮喘)患者T淋巴细胞增殖/凋亡功能和细胞因子合成功能的干预作用. 方法将T淋巴细胞分为四组正常对照组(A组)、哮喘空白组(B组)、哮喘NF-κB顺式诱骗ODN组(B1组)和无序哮喘ODN组(B2组).两种ODN分别经脂质体转染至后两组细胞.通过流式细胞术、四甲基偶氮唑盐微量比色法检测T淋巴细胞的凋亡和增殖;通过原位细胞杂交和酶联免疫吸附试验检测T淋巴细胞白细胞介素5(IL-5)mRNA和蛋白质的表达;通过逆转录-聚合酶链反应(PT-PCR)和Western blot检测T淋巴细胞诱导型一氧化氮合酶(iNOS)mRNA和蛋白质的表达. 结果 B1组T淋巴细胞增殖率为0.220±0.020, 与B组(0.340±0.030)比较差异有显著性(P＜0.05);凋亡率为(10.8±1.3)%, 与B组[(8.1±1.2)%]比较差异也有显著性(P＜0.05); 同时B1组IL-5 mRNA和蛋白质的表达分别为21±4、24±4,与B组(33±4、54±10)比较,差异有显著性(P＜0.05);B1组iNOS mRNA和蛋白质的表达分别为0.33±0.05、782±117,与B组(0.75±0.13、1 185±230)比较,差异有显著性(P＜0.05).而B2组与B组比较,上述指标的差异无显著性(P均>0.05). 结论 NF-κB顺式诱骗元件能使哮喘患者异常增殖的T淋巴细胞增殖减少、凋亡增多,使其过度分泌的炎性因子合成减少.有进一步探讨其作为新的哮喘基因治疗手段的价值.     

吸入糖皮质激素对哮喘患者诱导痰炎性细胞蛋白激酶Cα表达及白细胞介素-5的影响

目的　观察哮喘患者气道炎性细胞中蛋白激酶C(PKCα)的表达和白细胞介素 (IL) 5的水平 ,及吸入糖皮质激素 (以下简称激素 )对其的影响。方法　 2 9例哮喘患者分为激素治疗 2周组 ( 14例 )和激素治疗 4周组 ( 15例 ) ,治疗前后行诱导痰和肺功能检查。免疫组化 (SP法 )测PKCα在诱导痰炎性细胞中的表达 ,ELISA测诱导痰上清中IL 5的含量。结果　哮喘患者治疗前诱导痰中嗜酸性粒细胞 (EOS)与淋巴细胞相对计数、炎性细胞中PKCα阳性表达率与IL 5含量均高于健康对照组 (P <0 0 1) ,治疗后均明显下降 (P <0 0 1) ,但仍高于健康对照组 (P <0 0 1) ,激素治疗 2周组与激素治疗 4周组间无明显差异 (P >0 0 5 )。第 1秒钟用力呼气容积 (FEV1)占预计值的百分比与炎性细胞中PKCα的阳性表达率、IL 5浓度、EOS相对计数呈负相关 (r =- 0 4 2 3,P <0 0 5 ;r =- 0 6 6 4 ,P <0 0 1;r =-0 5 78,P <0 0 1) ;IL 5浓度与炎性细胞中PKCα的阳性表达率、EOS相对计数呈正相关 (r =0 6 2 3,P<0 0 1;r=0 75 8,P <0 0 1)。结论　PKCα信号途径可能为哮喘气道炎症发生的重要机制之一 ;吸入激素可明显降低气道IL 5的含量和炎性细胞PKCα阳性表达率 ,但短期内不能使气道炎症完全恢复正常     

Shift toward T helper 1 cytokines by type II collagen-reactive T cells in patients with rheumatoid arthritis

OBJECTIVE: To investigate the impact of type II collagen (CII)-reactive T cells on the Th1/Th2 cytokine balance in patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII were examined in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) by mixed lymphocyte culture. CII-reactive T cell lines were generated from the SFMC and PBMC. Interferon-gamma (IFNgamma), interleukin-12 (IL-12), and IL-4 were measured by enzyme-linked immunosorbent assay in the SF, sera, and culture supernatants of PBMC and SFMC that had been stimulated with CII. RESULTS: The frequency of CII-reactive T cells was higher in the PBMC from RA patients than in that from osteoarthritis patients and healthy control subjects. In RA patients, CII-reactive T cells were more prevalent in SFMC than in PBMC. The mean level of IFNgamma and the ratio of IFNgamma to IL-4 were significantly higher in the culture supernatants of T cells stimulated with CII; these differences were more prominent in SFMC. Levels of IL-12 in the culture supernatants of SFMC and PBMC stimulated with CII were significantly higher than those in unstimulated supernatants. T cell responsiveness correlated well with the level of type 1 cytokines in culture supernatants from RA T cells stimulated with CII. In the CII-reactive cell lines, the increased production of IFNgamma was consistent with clonal expansion. CONCLUSION: CII-reactive T cells are more abundant in SFMC than in PBMC and are strongly associated with a shift toward Thl cytokine in the inflamed joints of RA patients. Our results suggest that a skewing toward type 1 cytokines by CII-reactive T cells may play an important role in the chronic inflammatory process of RA.