雷公藤对致敏小鼠T细胞IL-5 mRNA表达及转录因子GATA-3活性的抑制作用

探讨雷公藤内酯醇 (TP )对致敏小鼠T淋巴细胞IL 5mRNA表达的影响及其机制。采用卵蛋白 (OVA )致敏的方法建立模型 ;运用原位杂交染色法 (ISH )观察TP对T淋巴细胞IL 5mRNA表达的影响 ;通过凝胶电泳迁移率实验 (EMSA )对CD4+T淋巴细胞核转录因子GATA 3的DNA结合活性进行检测 ,同时就TP的作用与地塞米松 (DM )相比较。结果表明致敏小鼠T淋巴细胞IL 5mRNA表达显著高于正常对照组 (P <0 0 1) ,经TP、DM处理后 ,其IL 5mRNA表达显著低于致敏组(P <0 0 1)。致敏小鼠CD4+ T淋巴细胞体外经伴刀豆蛋白A (ConA )刺激后 ,GATA 3的DNA结合活性与正常对照组比较显著增强 ,并呈时间依赖关系 ,经TP、DM处理后 ,GATA 3的DNA结合活性显著减弱。TP抑制IL 5基因转录的分子机制可能与其抑制GATA 3的DNA结合活性有关。     

雷公藤抑制致敏小鼠CD4~ T细胞核因子GATA_3、NFAT的活性

目的探讨致敏小鼠CD4`~ T淋巴细胞核转录因子GATA_3、NFAT活性的变化及雷公藤内酯醇(TP)的作用。方法采用卵蛋白(OVA)致敏的方法建立模型;运用panni ng法分离CD4~ T淋巴细胞;通过凝胶电泳迁移率实验(EMSA)对CD4~ T 淋巴细胞核因子GATA_3、NFAT的DNA结合活性及TP的作用进行检测,同时就TP的作用与地 塞米松(DM)相比较。结果正常小鼠CD4~ T 淋巴细胞核因子GATA_3、NFAT的活性很弱,致敏小鼠CD4~ T淋巴细胞体外经伴刀 豆蛋白A(ConA)刺激后,GATA_3、NFAT的活性与正常对照组比较显著增强,并呈时间依 赖关系。经TP、DM处理后,GATA_3、NFAT的活性显著减弱。结论 GATA_3和NFAT是调控Th2类细胞因子基因转录的重要核因子。TP、DM抑制Th 2类细胞因子基因转录的分子机制可能与其抑制GATA_3、NFAT的DNA结合活性有关。     

雷公藤抑制哮喘豚鼠模型IL-5、GM-CSF 表达以及核因子-κB的作用

目的 探讨雷公藤对哮喘豚鼠模型IL－5、GM－CSF表达的影响及其机制。方法 采用卵蛋白（OA）致敏复制哮喘豚鼠模型,用原位杂交（ISH）就雷公藤甲素（TP）对其肺组织和外周血单个核细胞（PBMC）IL－5、GM－CSF mRNA表达的影响进行检测。并通过凝胶电泳迁移试验（EMSA）检测TP对伴刀豆蛋白（ConA)或佛波脂（PMA）刺激的人T细胞核因子－κB（NF-κB）的DNA结合活性的影响。结果 哮喘豚鼠肺组织和PBMC IL－5、GM－CSF mRNA表达显著高于正常组（P＜0．01）和TP处理组（P＜0．05或0．01）。ConA或PMA刺激可使T细胞NF－κB的DNA结合活性增强,活性增强的NF－κB为多克隆抗体P65和P50的异源二聚体,TP可以降低该NF－κB的DNA结合活性。结论 雷公藤通过抑制NF－κB的DNA结合活性,进而抑制IL－5、GM－CSF mRNA的转录,可能是其治疗哮喘的机制之一。     

不同Th1/Th2细胞免疫应答支气管肺泡灌洗液中细胞学的变化

目的 探讨不同Th细胞优势应答下支气管肺泡灌洗液（BALF）中的细胞学变化，了解Th1/Th2细胞免疫应答的细胞和分子机制。方法 用鸡卵清蛋白（OVA）致敏Wistar大鼠（每组10只），制作致敏大鼠哮喘模型；用“冻干BCG”皮内注射制作BCG免疫大鼠模型。收集BALF并做HE染色，进行细胞分类计数。采用流式细胞术，测定BALF中，CD2^ ,CD28^ 及γδTCR^ T细胞占总淋巴细胞的百分率及平均荧光密度（MIF）。用原位杂交法，检测肺组织中IL－4mRNA的IFN－γmRNA的表达。用ELISA法检测血清IL－4和IFN－γ的浓度。结果 哮喘组BALF中淋巴细胞，嗜酸性粒细胞（EOS），浆细胞和中性粒细胞的总数，均显著多于正常组（P＜0．01）；BCG免疫组BALF中，淋巴细胞和巨噬细胞的总数也显著高于正常组（P＜0．001）。哮喘组BALF中，CD2^ T 细胞的明显增加。但哮喘组CD2^ T细胞的F1显著高于正常组及BCG组（P＜0．05）；BCG组BALF中，CD^2 T细胞的百分率与正常组相比产无显著差异（P＞0．05），其CD2^ T细胞的MFI显著高于正常组(P＜0．05）。哮喘组和BCG组BALF中，CD28^ 细胞占淋巴细胞的百分率显著多于正常组（P＜0．01）；BCG组CD28^ 细胞的MFI高于哮喘组（P＜0．01）。两组的CD28^ 细胞的MF1均显著多于正常组（P＜0．05）。哮喘组和BCG组BALF中，γδTCR^ 细胞占淋巴细胞的百分率显著高于正常组（P＜0．01）。结论 支气管哮喘患者Th2细胞的优势应答，与BALF中的B细胞，EOS，浆细胞和中性粒细胞等APC数的增加及T细胞上CD2的高表达有关；而BCG免疫组中的Th1细胞的优势应答与BALF中巨噬细胞，T细胞增加及T细胞上CD28的高表达有关。γδT细胞可能存在Th1/Th2细胞免疫模式，既参与哮喘免疫也参与BCG免疫过程，可能是调节Th0细胞分化的重要始动细胞。     

系统性红斑狼疮患者淋巴细胞CD69表达及细胞因子水平研究

目的：通过检测系统性红斑狼疮患者外周血中淋巴细胞CD69^ 表达及CD69＋细胞分泌细胞因子的水平，探讨淋巴细胞活化和活化淋巴细胞因子的表达与系统性红斑狼疮的发病关系。方法：利用流式细胞分析法对系统性红斑狼疮患者动期13例、静止期23例和健康志愿者15例外周血中淋巴细胞的CD69^ 表达及其细胞内细胞因子（IL－2、TNFα）进行分析。结果：活动期和静止期系统性红斑狼疮患者淋巴细胞CD69^ 的表达均显著高于正常组（P＜0.05），系统性红斑狼患者淋巴细胞CD69^ 表达与SLEDAI具有明显的相关性（P＜0.05），而且活动期系统性红斑狼疮患者活化淋巴细胞内的细胞因子IL－2、TNFα表达较正常组显著升高（P＜0.05）。结论：系统性红斑狼疮患者体内存在淋巴细胞早期活化现象，与SLE发病程度相关。活化的淋巴细胞内细胞因子IL－2、TNFα呈高表达状态。     

再生障碍性贫血患者淋巴细胞表型变化

目的：研究再生障碍性贫血（AA）患者骨髓（BM）及外周血（PB）淋巴细胞及其活化相关分子的表达及临床意义。方法：采用单色和双色免疫荧光标记法，流式细胞仪分析AA患者的BM和PB中淋巴细胞膜分子的表达。结果：AA患者BM和BP中CD8^ 细胞增加，CD4／CD8比例下降，BM在CD25^ 细胞和HLA－DR^ 细胞增多，急性AA增加尤为显著（P＜0．01），BM中CD16^ 或CD56^ 细胞也明显增多（P＜0．05），双标记分析提示T细胞主要为CD8^ 细胞：急性AA患者CD8^ -CD25^ 细胞显著增多（P＜0．01），AA患者BM中淋巴细胞活化相关分子表达增多，尤其4－1BB^ ,CD95L^ 和CD40L＋细胞显著增多（P＜0．01），结论：AA患者BM中淋巴细胞活化相关膜分子增多，是AA免疫功能异常及最终导致造血功能衰竭的原因之一。     

雷公藤甲素对哮喘豚鼠肺内嗜酸性粒细胞IL-5等受体表达的影响

目的 观察哮喘豚鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF)中不同密度嗜酸细胞（Eos)上白介素－5受体α（IL－5Rα）、IL－3Rα、粒细胞－巨噬细胞集落刺激因子受体α（GM－CSFRα）及共同β链受体（βcR)mRNA表达及雷公藤甲素（TP）对它们的影响，探讨TP促进哮喘Eos凋亡的机制。方法 健康豚鼠18只随机分为正常组、哮喘组、TP组。以卵蛋白致敏激发制作豚鼠哮喘模型，分离BALF中的低密度Eos(HEos)及正常密度Eos(NEos)，TUNEL法检测细胞凋亡，原位杂交检测各受体mRNA表达，RT－PCR法检测Eos IL-5Rα、IL－3Rα相对含量。结果 哮喘豚鼠Eos凋亡明显减少，而细胞数明显高于正常组。用TP 24h后不同密度Eos数量明显低于哮喘组（P＜0．01），以HEos下降为明显（P＜0．05）；TP组Eos凋亡明显高于哮喘组（P＜0．01）。哮喘豚鼠Eos表达α受体mRNA明显低于正常组，而βcR表达明显增高；TP处理组Eos各α受体mRNA表达均明显增加，βcR表达明显下降。RT－PCR检测显示，TP组IL－5Rα、IL－3RαmRNA显著增加。结论 哮喘Eos存在凋亡抑制；TP可通过促进IL－5、IL－3、GMPCSF受体各自α链表达，抑制这3种受体的共同β链表达，减少其生物学功能，促进Eos凋亡。     

HIV-1感染者淋巴细胞活化与第二受体表达的研究

目的：了解HIV－1感染者体内淋巴细胞的活化情况及表达第二受体CCR5、CXCR4的淋巴细胞活化状态，分析这些指标与疾病严重程度的关系，探讨HIV感染的免疫基础。方法：用三色标记法流式细胞术检测24例HIV－1感染者及13例健康对照的抗凝血标本，分析活化标志物HLA－DR及第二受体CCR5、CXCR4的表达等指标。结果：HIV－1感染者CD8^ T淋巴细胞的HLA－DR表达高于健康对照（P＜0．001）；HIV－1感染者表达CCR5、CXCR4的CD8^ T淋巴细胞活化明显高于健康对照（P＜0．001）；表达CCR5CD4^ 、CD8^ T淋巴细胞与表达CXCR4相比HL－DR表达均明显增高（P＜0．001）；CD4^ 、CD8^ T淋巴细胞的活化状态与CD4百分率的变化明显关系。结论：HIV－1感染者CD8^ T淋巴细胞及表达不同第二受体的CD8^ T淋巴细胞活化程度明显增高，活化程度与疾病进程相关。     

DNAM-1在系统性红斑狼疮患者T淋巴细胞的表达研究

目的：研究DNAM－1在SLE患者外周血T淋巴细胞亚群上的表达，以阐明DNAM－1抗原在SLE患者体内活化作用以及与SLE发病的关系。方法：31例SLE患者和30例健康志愿者外周血单个核细胞，在PHA刺激培养72小时后，三色荧光标记的单克隆抗体染色，利用流式细胞仪测定T淋巴细胞亚群膜表面DNAM－1抗原的表达。同时检测SLE患者抗dsDNA抗体、C3和C4补体，疾病活动性用SLEDAI记分。结果：SLE患者CD4^＋、CD8^＋淋巴细胞上DNAM－1表达率均高于正常对照组（P＜0．01）；活动期SLE组CD4^＋、CD8^＋细胞上DNAM－1表达高于正常对照组和静止期SLE组（P＜0．01），而静止期SLE组与正常对照组无明显性差异（P＞0．05）；SLE患者CD8^＋细胞DNAM－1表达与SLEDAI、抗dsDNA抗体之间呈显著正相关（P＜0．001），与C3和C4补体水平呈明显负相关（P＜0．05），CD4^＋细胞DNAM－1表达与SLEDAI、抗dsDNA抗体、C3和C4补体之间无明显相关性（P＞0．05）。结论：SLE患者内存在T细胞亚群异常活化；活动期SLE患者T淋巴细胞亚群DNA－1表达增高；SLE患者CD8^＋细胞DNAM－1表达异常与SLEDAI、抗dsDNA抗体、C3和C4补体之间有明显相关，CD8^＋细胞活化程度可能与SLE疾病严重程度有关，故DNAM－1可能参与了SLE的免疫发病机理。     

休克期大面积切痂对严重烧伤大鼠细胞免疫功能影响的研究

目的 观察休克期大面积切痂对严重烧伤大鼠细胞免疫功能的影响，探索改善烧伤后机体免疫功能紊乱的有效方法。方法 将大鼠分成休克期切痂组（A组）、常规切痂组（B组）和正常对照组（C组）。A、B组造成30％TBSAⅢ度烫伤，C组不烫伤。A组伤后第6h、B组伤后第4d切痂，并于伤后第1、5、9d各活杀10只，取材送检，观察其免疫指标的变化。结果 （1）A、B组与C组比较：A、B组烫伤大鼠各时相点CD3^＋T细胞变化不大（P〉0．05），但CD4^＋T细胞、CD4^＋／CD8^＋比值明显下降、CD8^＋T细胞增高（P〈0．05或P〈0．01）。NK细胞活性明显下降（P〈0．05或P〈0.01），外周血CD25^＋T淋巴细胞表达及经活化后脾脏CD25^＋T淋巴细胞表达明显下降（P〈0．05或P〈0．01）。（2）A组与B组比较：A组CD4^＋T细胞、CD4^＋／CD8^＋比值明显升高、CD8^＋T细胞降低（P〈0．05或P〈0．01），NK细胞活性明显升高（P〈0．05或P〈0．01），外周血CD25^＋T淋巴细胞表达及经活化后脾脏CD25^＋T淋巴细胞表达均明显升高（P〈0．05或P〈0．01）。结论 （1）大鼠烫伤后细胞免疫状况发生了明显变化。（2）休克期切痂可以改善烫伤大鼠T淋巴细胞亚群分布，提高NK细胞活性，增加外周血CD25^＋T淋巴细胞的表达。提高经活化后脾脏CD25^＋T淋巴细胞数。从而改善烫伤大鼠伤后机体的细胞免疫功能。     

雷公藤内酯醇对致敏大鼠淋巴细胞凋亡的影响

目的 探讨雷公藤内酯醇（TP）体内外对致敏大鼠淋巴细胞淋巴细胞凋亡的影响。方法 采用卵蛋白（OVA）致敏并反复刺激建立过敏性气道炎症模型,24只SD大鼠随机分为正常组、阳性对照组和TP处理组,每组8只。用TUNEL原位末端标记法,DNA电泳及电镜等方法,观察体内外TP对致敏大鼠淋巴细胞凋亡的影响及机制。结果 致敏大鼠BALF中嗜酸性粒细胞（Eos)、淋巴细胞均较正常组明显增高（P＜0．05）。体内应用TP可减少致敏大鼠BALF中Eos、淋巴细胞数目,同时可增加其BALF中淋巴细胞凋亡百分率。体外实际显示,不同剂量TP呈剂量依赖性（10^-7-10^5g/ml）的促进OVA抗原刺激的脾淋巴细胞凋亡,该效应随作用时间凋亡,可能是其抗炎机制之一,并可能是通过Fas/FasL途径发挥作用;同时TP可明显增加DXM的促淋细胞凋亡作用。为阐明TP对抗哮喘气道炎症的作用机制和探讨激素依赖性哮喘治疗的新途径提供了有意义的实验资料。     

Coxsackievirus B3 induction of NFAT: requirement for myocarditis susceptibility

Ultraviolet (u.v.) inactivated coxsackievirus B3 (CVB3) induces rapid calcium flux in naïve BALB/c CD4+ T cells. CD4+ cells lacking decay accelerating factor (DAF−/−) show little calcium flux indicating that virus cross-linking of this virus receptor protein is necessary for calcium signaling in CVB3 infection. Interaction of CVB3 with CD4+ cells also activates NFAT DNA binding. To show that NFAT activation is crucial to CVB3 induced disease, wild-type mice and transgenic mice expressing dominant-negative NFAT (dnNFAT) mutant in T cells were infected and evaluated for myocarditis and pancreatitis 7 days later. Inhibition of NFAT in T cells prevented myocarditis but had no effect on pancreatitis. Virus titers in pancreas were equivalent in wild-type and dnNFAT animals but cardiac virus titers were increased in dnNFAT mice. Interferon-gamma (IFNγ) expression was reduced in both CD4+ and Vγ4+ T cells from dnNFAT mice compared to controls. FasL expression by Vγ4+ cells was also suppressed. Inhibition of FasL expression by Vγ4+ cells is consistent with myocarditis protection in dnNFAT mice.     

羧胺三唑通过抑制NFAT2核转运降低小鼠CD8^+T细胞中程序性死亡受体1的表达

目的研究羧胺三唑(CAI)对CD8+T细胞的作用,并探索其增强活化的T细胞杀伤作用的关键机制。方法用免疫磁珠分选出小鼠CD8+T细胞,分为对照组、CAI组、ZK756326(Ca2+激活剂)组以及CAI+ZK756326组。流式细胞计量术检测细胞内游离钙离子水平;酶联免疫吸附法检测细胞内钙调磷酸酶(CaN)的表达;免疫荧光染色检测细胞活化T细胞核因子2(NFAT2)核转运;染色质免疫共沉淀-qPCR检测细胞中NFAT2调控程序性死亡受体1(PD-1)表达。分离小鼠脾脏细胞毒性T淋巴细胞(CTLs),流式细胞计量术检测其中CD8+T细胞PD-1的表达。结果CAI组显著降低CD8+T细胞内Ca2+浓度(P<0.001),CAI+ZK756326组胞内Ca2+浓度有所升高(P<0.01);CAI显著降低CD8+T细胞中钙调磷酸酶的含量(P<0.001);CAI可显著抑制NFAT2核转运(P<0.001),并使NFAT2依赖的PD-1转录进程显著降低(P<0.001);CAI使小鼠脾脏CTLs细胞中PD-1+CD8+T细胞比例显著减少(P<0.001)。结论CAI通过抑制CD8+T细胞内钙离子水平以及钙调磷酸酶的表达抑制NFAT2核转运,从而降低CD8+T细胞PD-1的表达,进一步产生免疫治疗干预作用。     

Antigen‐specific airway IL‐33 production depends on FcγR‐mediated incorporation of the antigen by alveolar macrophages in sensitized mice

Although interleukin (IL)‐33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen‐specific IL‐33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra‐tracheal administration of ovalbumin (OVA) evoked increases in IL‐33 and IL‐33 mRNA in the lungs of both non‐sensitized and OVA‐sensitized mice, and the increases in the sensitized mice were significantly higher than in the non‐sensitized mice. However, intra‐tracheal administration of bovine serum albumin did not increase the IL‐33 level in the OVA‐sensitized mice. Depletion of neither mast cells/basophils nor CD4⁺ cells abolished the OVA‐induced IL‐33 production in sensitized mice, suggesting that the antigen recognition leading to the IL‐33 production was not related with either antigen‐specific IgE‐bearing mast cells/basophils or memory CD4⁺ Th2 cells. When a fluorogenic substrate‐labelled OVA (DQ‐OVA) was intra‐tracheally administered, the lung cells of sensitized mice incorporated more DQ‐OVA than those of non‐sensitized mice. The lung cells incorporating DQ‐OVA included B‐cells and alveolar macrophages. The allergic IL‐33 production was significantly reduced by treatment with anti‐FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL‐33 production, whereas depletion of B‐cells by anti‐CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen‐specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL‐33 production in sensitized mice. The mechanisms underlying the antigen‐specific IL‐33 production may aid in development of new pharmacotherapies.     

EGF receptor activation during allergic sensitization affects IL‐6‐induced T‐cell influx to airways in a rat model of asthma

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA‐specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4⁺ T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL‐6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA‐specific IgE and IL‐4 mRNA expression in CD4⁺ T cells were not affected by AG1478. BALF from OVA‐sensitized/challenged rats induced CD4⁺ T‐cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL‐6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4⁺ T cells to airways, mainly mediated through IL‐6.     

The effects of triptolide on airway remodelling and transforming growth factor-β₁/Smad signalling pathway in ovalbumin-sensitized mice

Airway remodelling contributes to increased morbidity and mortality in asthma. We have reported that triptolide, the major component responsible for the immunosuppressive and anti‐inflammatory effects of Tripterygium wilfordii Hook F, inhibited pulmonary inflammation in patients with steroid‐resistant asthma. In the present study, we investigated whether triptolide inhibits airway remodelling in a mouse asthma model and observed the effects of triptolide on the transforming growth factor‐β₁ (TGF‐β₁)/Smad pathway in ovalbumin (OVA) ‐sensitized mice. BALB/c mice were sensitized to intraperitoneal OVA followed by repetitive OVA challenge for 8 weeks. Treatments included triptolide (40 μg/kg) and dexamethasone (2 mg/kg). The area of bronchial airway (WAt/basement membrane perimeter) and smooth muscle (WAm/basement membrane perimeter), mucus index and collagen area were assessed 24 hr after the final OVA challenge. Levels of TGF‐β₁ were assessed by immunohistology and ELISA, levels of TGF‐β₁ mRNA were measured by RT‐PCR, and levels of pSmad2/3 and Smad7 were assessed by Western blot. Triptolide and dexamethasone significantly reduced allergen‐induced increases in the thickness of bronchial airway and smooth muscle, mucous gland hypertrophy, goblet cell hyperplasia and collagen deposition. Levels of lung TGF‐β₁, TGF‐β₁ mRNA and pSmad2/3 were significantly reduced in mice treated with triptolide and dexamethasone, and this was associated with a significant increase in levels of Smad7. Triptolide may function as an inhibitor of asthma airway remodelling. It may be a potential drug for the treatment of patients with a severe asthma airway.