IL-17和RORγt在类风湿关节炎CD4+T细胞中的表达及临床意义

目的探讨类风湿关节炎(RA)患者外周血CD4+ T细胞中白细胞介素(IL)-17、孤核受体(RORγt)表达及意义。方法 35例RA患者分为病情活动组(A组,20例)和RA病情稳定组(B组,15例);另设健康人作对照(C组,15例)。取外周血单个核细胞(PBMC),免疫磁珠阴选CD4+ T细胞,通过流式细胞术(FCM)检测CD4+ T细胞内IL-17蛋白水平;非特异性刺激剂(A-CD3、A-CD28)刺激CD4+ T细胞后,RT-PCR检测IL-17、RORγt mRNA水平。结果与B组和C组比较,A组IL-17蛋白水平和IL-17、RORγt mRNA表达均显著增高(P<0.01),且B组各指标也高于C组(P<0.05)。A-CD3/A-CD28刺激CD4+ T细胞12h后,IL-17、RORγt mRNA表达水平较刺激24h明显增加(P<0.01),且两组各指标亦均高于未刺激组(P<0.05)。结论 RA患者外周血CD4+ T细胞中IL-17、RORγt mRNA高表达,这可能与RA病情活动相关。     

白细胞介素15对儿童骨髓增生异常综合征造血前体细胞caspase-3 mRNA表达的影响

目的:探讨白细胞介素15(IL-15)对骨髓增生异常综合征(MDS)造血细胞caspase-3 mRNA表达的影响,以探讨IL-15抑制MDS CD34+造血干/祖细胞凋亡的可能机制.方法:采用吸附单克隆抗体的免疫磁珠分离系统分离纯化18例MDS患者骨髓CD34+细胞,采用逆转录-聚合酶链式反应(RT-PCR)检测caspase-3 mRNA表达水平,观察IL-15对它们的影响.结果:发现IL-15下调体外培养的MDS造血前体细胞cpp32基因mRNA的表达在0～100 ng/mL范围内呈剂量依赖性,其差异具有统计学意义;从d 1～d 8,随着IL-15作用时间延长,下调caspase-3基因mRNA表达的能力越强,其差异具有统计学意义.结论:IL-15可呈时间与剂量依赖性的下调体外培养的MDS造血前体细胞caspase-3基因mRNA的表达.IL-15下调MDS CD34+造血干/祖细胞caspase-3 mRNA的表达,可能为其抑制该细胞凋亡的作用机制之一.     

银杏外种皮多糖对环磷酰胺诱导的免疫抑制小鼠免疫反应的调节作用

目的研究银杏外种皮多糖(GBEP)对环磷酰胺(CPA)所致免疫抑制模型小鼠细胞免疫及体液免疫的调节作用。方法将ICR小鼠随机分为对照组、模型组及3个GBEP治疗组。模型组ipCPA40mg.kg-1,同时ig等体积生理盐水(NS);治疗组分别igGBEP50,100和200mg.kg-1,同时ipCPA40mg.kg-1;对照组分别ig和ip等体积NS。每日1次,连续给药5d。于d6采用葡萄球菌A蛋白花环法测定小鼠外周血T淋巴细胞亚群,采用双抗体夹心ABC-ELISA法测定小鼠血清可溶性白细胞介素-2受体(sIL-2R)含量,采用MTT法检测小鼠脾脏T细胞IL-2活性,采用免疫比浊法测定小鼠血清免疫球蛋白(Ig)的含量。结果模型组小鼠外周血CD3+T细胞及CD4+T细胞百分率下降,血清中sIL-2R含量升高,脾脏T细胞产生IL-2被抑制,血清中IgM,IgG和IgA含量减少。GBEP3个剂量均可提高模型小鼠外周血CD3+和CD4+T细胞百分率,降低血清sIL-2R含量,促进脾脏T细胞IL-2活性以及血清IgM,IgG和IgA的生成。结论GBEP可调节CPA抑制的小鼠细胞免疫及体液免疫功能。     

抗CD47抗体Hu5F9真核表达载体构建与表达

目的 在CHO-S细胞中表达抗白细胞分化抗原47(CD47)抗体Hu5F9,并对抗体纯化后进行鉴定。方法 将编码Hu5F9抗体轻、重链基因构建至真核表达载体,瞬转CHO-S细胞进行表达,取细胞上清检测后利用其人源Fc标签纯化抗体蛋白,并进行Western blot鉴定。结果 经双酶切及基因测序验证Hu5F9载体构建成功,细胞上清经SDS-PAGE检测,可见抗体轻链、重链和完整抗体条带,抗体纯化后Western blot鉴定结果显示条带大小与预期一致。结论 在CHO-S细胞中成功表达了抗CD47抗体Hu5F9,为后续的药物研发提供研究依据。     

重组Exendin-4-胰高血糖素样肽1/IgG4(Fc)融合蛋白的表达和活性研究

目的  构建表达长效胰高血糖素样肽1（glucagon-like peptide 1,GLP-1）受体激动剂重组Exendin-4-GLP-1/IgG4(Fc)融合蛋白的质粒载体,并研究该融合蛋白的活性。方法  将编码Exendin-4-GLP-1/IgG4(Fc)的重组基因插入表达载体pOptiVEC™-TOPO^®来构建重组质粒Exendin-4-GLP-1/IgG4(Fc)-pOptiVEC™-TOPO^®。将构建的重组质粒转染CHO/DG44细胞并收获表达产物后,分别用亲和层析法和免疫印迹法对表达产物进行纯化和检测。通过胰岛素释放实验确认重组融合蛋白对INS-1细胞胰岛素分泌的影响,同时在CD1小鼠中研究该融合蛋白对血糖的调节作用。结果  转染重组质粒的CHO/DG44细胞可成功表达Exendin-4-GLP-1/IgG4(Fc)。蛋白质印迹法检测显示,纯化的表达产物的相对分子质量（M_r）与预期相符（重组融合蛋白单体和二聚体的M_r分别约为35 000和70 000）。胰岛素释放实验表明,在葡萄糖浓度恒定的情况下INS-1细胞分泌的胰岛素量随重组融合蛋白浓度的升高而增加。CD1小鼠实验显示,重组融合蛋白对链脲佐菌素诱导的糖尿病小鼠的血糖具有调节作用,Exendin-4-GLP-1/IgG4(Fc)处理的糖尿病小鼠的血糖明显低于对照糖尿病小鼠（F=3194,P＜0.01）。结论  Exendin-4-GLP-1/IgG4(Fc)具有天然GLP-1的活性,可作为GLP-1受体激动剂用于2型糖尿病的治疗。     

rhIL-12联合HBsAg刺激健康人外周血单核细胞对IL-12R的影响

目的确认重组人白细胞介素12(rhIL-12)联合乙型肝炎表面抗原(HBsAg)可增强健康人外周血单核细胞(PBMC)产生特异性细胞免疫应答,并分析其细胞来源,及对IL-12R的影响,探讨hIL-12联合HBsAg作为治疗型乙型肝炎疫苗的应用前景。方法 8名健康人PBMC分别与rhIL-12(1 ng/mL)、rHBsAg(500 ng/mL)和rhIL-12(1ng/mL)+rHBsAg(500 ng/mL)体外孵育,采用流式细胞术检测CD4+IFN-γ(γ-干扰素)+T细胞、CD8+IFN-γ+T细胞及CD56+IFN-γ+NK细胞百分比,并测定表达IL-12Rβ1、IL-12Rβ2的细胞百分比。结果 rhIL-12与rHBsAg联合组CD8+IFN-γ+T细胞及CD56+IFN-γ+NK细胞百分比均较对照组明显升高,且有非常显著差异(P<0.01),联合组CD8+IL-12Rβ1和CD56+IL-12Rβ1与单用rhIL-12或rHBsAg组比较均显著升高。结论提示rhIL-12联合rHBsAg诱生的IFN-γ主要来自CD56+NK细胞、CD8+T细胞,其次是CD4+T细胞;rhIL-12和rHBsAg单独均能上调健康人PBMCs表面IL-12R的表达,rhIL-12与rHBsAg联合具有增强作用。     

肌肉注射白介素-10质粒DNA预防小鼠自身免疫性糖尿病

目的:研究编码白介素-10(IL-10)质粒DNA对由多次、低剂量链脲佐菌素(STZ)诱发的自身免疫性糖尿病小鼠的作用。方法:连续5天将STZ(每次40 mg/kg)腹腔注射入小鼠体内,于第1、14天将100 mg表达人IL-10-pcDNA3质粒(IL-10处理组)或pcDNA3质粒(对照组)注射入骨骼肌内。测定小鼠血糖水平。第28天处死小鼠,检测小鼠血清干扰素-γ(IFN-γ)水平、胰腺IL 1β和TNF-α mRNA表达,测定脾CD4~+和CD8~+淋巴细胞的数量,同时进行胰腺组织学检查。结果:IL-10质粒DNA注射后,骨骼肌有IL-10 mRNA的持久表达,血浆IL-10水平明显升高,而且对迟发性超敏反应具抑制作用。IL-10处理组,在14、21和28天小鼠血糖显著降低,在21、28天时糖尿病发病率分别为33.3％和40.0％,显著低于对照组(P<0.05);胰腺IL-1β和TNF-αmRNA表达、血清IFN-γ水平,以及脾CD4~+和CD8~+淋巴细胞数量均低于对照组,胰岛炎症程度也明显轻于对照组(P<0.01)。结论:IL-10基因治疗能减轻实验性自身免疫性糖尿病小鼠 的胰岛炎症,降低糖尿病发生的机率。     

IL-18及IL-18抗体对刀豆蛋白A所致急性肝损伤的作用

目的 探讨白细胞介素18(IL-18)及IL-18抗体对刀豆蛋白A所致急性肝损伤的预防作用及机理.方法 昆明小鼠57只,随机分为:正常对照组、兔IgG组、刀豆蛋白A组和IL-18及IL-18抗体组,尾静脉分别注射生理盐水、兔IgG、刀豆蛋白A、IL-18及IL-18抗体.在第4、8、12h,采用眼眶采血法,留取血清,ELISA法检测肿瘤坏死因子-α(TNF-α),干扰素因子-γ(IFN-γ),并留取肝组织行HE染色计算炎症活动度.结果 各组实验结束时,非正常死亡数各组比较有显著性差异(P＜0.05).炎症活动度评分与同期IL-18组和刀豆蛋白A组比较有显著性差异(P＜0.05).TNF-α、IFN-γ的检测也显示IL-18组明显增高,而予IL-18抗体干扰的小鼠血清TNF-α、IFN-γ表达下降.结论 IL-18可以增强刀豆蛋白A诱导的急性肝损伤的炎症作用,而IL-18抗体可以阻断这种作用.其机理可能是通过上调和抑制TNF-α、IFN-γ的表达.     

单纯疱疹病毒性角膜炎淋巴细胞活化状态与γ-干扰素、白细胞介素2水平关系的研究

目的检测复发性单纯疱疹病毒性角膜炎(HSK)患者外周血淋巴细胞亚群及淋巴细胞CD4/HLA-DR+、CD8+/HLA-DR+的表达,测定HSK患者血清中γ-干扰素(IFN-γ)、白细胞介素2(IL-2)水平,研究HSK患者外周血淋巴细胞活化状态与血清中IFN-γ、IL-2水平的关系,探寻HSK复发的分子免疫机理,为临床治疗提供新思路。方法收集复发性单纯疱疹病毒性角膜炎病例22例,设置体检合格的健康人为正常对照组。取患者及正常对照组外周血,应用流式细胞技术对淋巴细胞亚群、淋巴细胞CD4+/HLA-DR+、CD8+/ULA-DR+的表达进行检测;ELISA双抗体夹心法检测患者血清IFN-γ、IL-2水平。结果(1)复发性单疱病毒性角膜炎患者外周血CD3+、CD4+细胞降低,CD8+、CD19+、CD16+56+细胞无明显变化,CD4/CD8降低,CD4+/HLA-DR+降低,CD8+/HLA-DR+升高,血清IFN-γ、IL-2水平降低,与正常对照组比较,差异有显著性意义(P<0.05)。(2)HSK患者CD4+细胞、CD4+/HLA-DR+表达的降低与血清IFN-γ、IL-2水平的改变呈正相关。结论复发性单纯疱疹病毒性角膜炎患者CD4+细胞、CD4+/HLA-DR+表达降低;IFN-γ、IL-2水平降低;CD4+细胞、CD4+/HLA-DR+、IFN-γ、IL-2与单纯疱疹病毒性角膜炎病情的发展、复发关系密切。     

Pim-1激酶对小鼠CD4+ T细胞分化的影响

探讨Pim-1 激酶对小鼠脾脏naive CD4+ T 细胞分化的影响。方法　采用不同的细胞因子诱导naive CD4+ T 细胞分化,PIM-Inh 阻断PIM-1 激酶,通过Western blot 技术检测小鼠脾脏naive CD4+ T 细胞分化过程中Tbet、GATA3、FOXP3、RORγt 蛋白的表达,ELISA 检测诱导分化的CD4+ T 细胞上清液中细胞因子IL-4、TGF-β 、IFN-γ、IL-17 的表达。结果　诱导分化的naive CD4+ T 细胞Tbet、GATA3、FOXP3、RORγt 蛋白的表达及上清液中细胞因子IL-4、TGF-β 、IFN-γ、IL-17 的表达均较空白对照组明显升高（P<0.05）,而PIM-Inh 可以抑制naive CD4+ T 细胞Tbet,RORγt 表达并降低上清液中IFN-γ、IL-17 的水平（P<0.05）。结论　Pim-1 激酶可能在naive CD4+ T 向Th1、Th17 分化过程中发挥了重要作用。     

玉屏风多糖对佐剂性关节炎大鼠T细胞亚群的影响

目的观察玉屏风多糖(Yupingfeng Polysaccharide,YPF-P)对佐剂性关节炎大鼠T细胞亚群的影响。方法采用弗氏完全佐剂(FCA)诱导大鼠AA模型。足容积法测量继发侧足肿胀度;MTT法检测刀豆蛋白(ConA)诱导的外周血淋巴细胞增殖反应;流式细胞仪检测大鼠外周血中CD4+和CD8+T细胞的变化;ELISA法检测大鼠外周血中干扰素γ(interferon-γ,IFN-γ)含量;放免法检测大鼠外周血中IL-4含量;RT-PCR法检测淋巴细胞IFN-γmRNA的表达。结果80、160mg.kg-1YPF-P给药能明显减轻关节肿胀度,不同程度地改善AA大鼠低下的外周血淋巴细胞增殖反应,明显降低CD4+/CD8+的相对比值。此外,80、160mg.kg-1YPF-P给药也能明显降低外周血IFN-γ含量和轻度提高IL-4含量,并能抑制IFN-γmRNA的表达。结论YPF-P能改善模型大鼠外周血T细胞亚群,纠正佐剂性关节炎模型大鼠Th1/Th2平衡的Th1偏移,这可能是减轻足肿胀的原因之一。     

华支睾吸虫病患者血清IL—2和T淋巴细胞亚群的测定研究

目的：探讨华支睾吸虫病患者的细胞免疫调节作用。方法：选择20例未经治疗的华支睾吸虫病患者（观察组）和20例健康对照组，检测血清中IL－2的活性以及CD4^-,CD8^-细胞含量，结果：观察组血清中IL－2含量较对照组明显降低，CD4^-1含量显著减少，CD8^-的含量增多，结论：华支睾吸虫病患者细胞免疫功能处于明显反应抑制状态。     

Different roles of IL-15 from IL-2 in differentiation and activation of human CD3+CD56+ NKT-like cells from cord blood in long term culture

The abundantly available source of stem cells and the low incidence of graft-versus-host disease (GVHD) made cord blood an attractive alternative source of hematopoietic stem cells for transplantation. Besides T cell and NK cell, NKT cell played an important role in low incidence of GVHD during allogeneic transplantation. IL-2 and IL-15 can stimulate T cell and NK cell proliferation, survival and activation in vitro. But they exhibited different effects on the GVHD during allogeneic transplantation. In this study, we explored the different effects of exogenous IL-2 and IL-15 on the expansion of CD3+CD56+ NKT-like cells by in vitro long term culture of cord blood mononuclear cells (CBMCs). The results showed that CD3+CD56+ NKT-like cells were derived from CD34-CD56- CBMCs and IL-2 improved CD3+CD56+ NKT-like cell expansion more strongly than IL-15. Interestingly, CD3+CD56+ NKT-like cells from IL-15-cocultured CBMCs had significantly lower apoptotic frequency and higher levels of activation markers (CD161, CD25, and IFN-gamma) than those from IL-2-cocultured CBMCs. The anti-apoptotic and activating effects of IL-15 on CD3+CD56+ NKT-like cells from CBMCs might possibly explain the pathogenic role of IL-15 in GVHD during allogeneic transplantation.     

Role of interleukin-21 in inflammation and allergy

Interleukin-21 (IL-21) is a newly described cytokine, produced by activated CD4+ T cells. Since the discovery in 2000, IL-21 has been the object of intensive research because of its homology to IL-2, IL-4 and IL-15, and its ability to modulate both innate and adaptive immune responses. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21 receptor (IL-21R) and the common gamma-chain, that is shared with IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15 receptors. IL-21R is originally described on T, B and NK cells, which is in accordance with the cell types that mostly respond to IL-21. Indeed, IL-21 augments the proliferation of CD4+ and CD8+ T lymphocytes and regulates the profile of cytokines secreted by these cells, drives the differentiation of B cells into memory cells and terminally differentiated plasma cells, and moreover, enhances the activity of natural killer cells. More recently, IL-21R has also been documented on non-immune cells, raising the possibility that IL-21 is an important mediator in the cross-talk between immune and non-immune cells. As discussed in this review, the potential role of IL-21 in immune-mediated and allergic diseases would seem to suggest that either disrupting or enhancing IL-21 signaling may be useful in specific clinical settings.     

Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo

BACKGROUND AND PURPOSE

Interleukin-15 (IL-15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL-15.

EXPERIMENTAL APPROACH

DISC0280 was characterized in a direct binding assay of IL-15 with IL-15 receptor α (IL-15Rα) and by its ability to alter IL-15 mediated proliferation of a range of cell lines (cytotoxic T lymphocyte line-2, M-07e, KIT225). A pharmacodynamic model injecting male C57/BL6 mice with IL-15 or IL-15/IL-15Rα, with or without DISC0280, and assessing changes in lymphocytic cell populations and serum cytokines was utilized.

KEY RESULTS

DISC0280 inhibited the binding of IL-15 to IL-15Rα and also potently inhibits IL-15 dependent proliferation of cells expressing IL-15Rα, shared interleukin 2/ interleukin 15 receptor β chain (IL-15Rβ) and common gamma chain (γ_c). DISC0280 also inhibited the IL-15 dependent proliferation of M-07e cells that only express IL-15Rβ/γ_c subunits. Human IL-15 injected into mice caused an increase in NK1.1⁺ and CD3⁺ cells in the spleen and peripheral blood and these effects were unexpectedly potentiated by giving DISC0280 with human IL-15. This increase in cells caused by DISC0280/IL-15 co-administration was greater than that observed when IL-15 was administered complexed with soluble IL-15Rα.

CONCLUSIONS AND IMPLICATIONS

The ability of DISC0280 to bind to the IL-15Rα-binding site on IL-15 allows trans-presentation of IL-15 by DISC0280 in vivo, similar to the trans-presentation by soluble IL-15Rα. DISC0280 may be therefore suitable as a clinical substitute for IL-15.     

哮喘患儿sIL-2R与细胞免疫功能检测的临床意义

目的探讨儿童支气管哮喘（简称哮喘）发作期淋巴细胞免疫功能和白细胞介素-2（IL-2）、可溶性白介素2受体（sIL-2R）检测的临床意义。方法应用流式细胞术和酶联免疫吸附试验（ELISA）方法对25例支气管哮喘患儿和21例支气管哮喘缓解期患儿以及20例正常儿童外周血T细胞亚群及血清IL-2和sIL-2R水平进行测定。结果支气管哮喘患儿发作期血清sIL-2R水平明显高于缓解期和对照组，缓解期sIL-2R水平仍高于对照组。IL-2水平明显低于缓解期和对照组。外周血总CD3^＋、CD4^＋、CD4^＋／CD8^＋细胞比值均降低，CD8^＋细胞增高。结论动态监测IL-2和sIL-2R水平含量，观察T细胞亚群变化可以判断哮喘发作的程度、指导治疗和评估预后。     

Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the<Emphasis Type="Italic">In Vitro</Emphasis> immune response in pristane-induced lupus mice

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.