CD4+CD25+调节性T细胞及Th17细胞与Graves病机制研究

目的：探讨CD4+CD25+调节性T细胞以及Th17细胞与Graves病的关系。方法：检测GD患者和对照组外周血单个核细胞CD4+CD25+Tregs、Th17细胞的数量,PBMC中TGF-β、IL-10和FoxP3 mRNA的表达水平以及血清TGF-β、IL-10和IL-17水平。结果：GD组外周血Th17数量高于对照组,血清中IL-17水平也高于对照组（P＜0.05）;而CD4+CD25+Tregs的数量低于对照组,而且TGF-β、FoxP3表达水平低于对照组（P＜0.05）;GD组血清中TGF-β、IL-10水平高于对照组（P＜0.05）。结论：CD4+CD25+Tregs数量的减少或功能障碍以及Th17细胞数量的升高,可能参与GD的发病过程。     

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。     

自身免疫性甲状腺炎小鼠发病中调节性T细胞、Th17细胞的变化

目的观察高碘诱发自身免疫性甲状腺炎(AIT)动物模型中CD4+CD25+调节性T细胞、Th17细胞的变化。方法选取NOD.H-2h4雌鼠饮0.005%碘化钠水,HE染色观察淋巴细胞浸润情况并进行甲状腺炎症程度评分;测定血清甲状腺球蛋白抗体(TgA b)水平;免疫荧光染色流式细胞仪分析CD4+CD25+Foxp3+调节性T细胞、Th17细胞比例的变化;实时定量RT-PCR检测Foxp3 mRNA、IL-17 mRNA、RORγt mRNA表达水平。结果 NOD.H-2h4小鼠高碘饮水后,甲状腺炎的发生率明显高于对照组,甲状腺组织有不同程度的淋巴细胞浸润,甲状腺相对重量及血清TgA b水平均较对照组小鼠明显升高(P<0.05)。脾细胞中CD4+CD25+Foxp3+调节性T细胞所占比例和Foxp3 mRNA表达量均较对照组明显降低(P<0.01);脾细胞中Th17细胞所占比例和IL-17 mRNA表达量、RORγt mRNA表达水平均较对照组明显升高(P<0.05)。结论 NOD.H-2h4小鼠在高碘诱导发生甲状腺炎时,脾脏CD4+CD25+调节性T细胞比例明显降低,Th17细胞比例明显升高。     

布鲁氏菌病患者外周血树突状细胞、Th1／Th2细胞因子含量的检测及意义

目的：观察布鲁氏菌病患者外周血树突状细胞表型、Th1／Th2细胞含量的检测及意义。方法选取诊治的布鲁氏菌病患者50例为病例组,另选取同期进行健康体检的正常人50例作为正常组。采用Real time-PCR测定2组Th1相关转录因子T细胞表达的T盒（T-bet）、GATA连接蛋白3（GATA-3）、维A酸相关核孤儿受体γt（RORγt）、叉头蛋白3（Foxp3）及Th1／Th2细胞因子肿瘤坏死因子α（TNF-α）、干扰素γ（IFN-γ）、白介素6（IL-6）、白介素10（IL-10）mRNA含量。酶联免疫吸附实验（ELISA）测定TNF-α、IFN-γ、IL-6、IL-10蛋白表达及补体C3、C4含量。流式细胞术测定树突状细胞表型、Th1、Th2及T淋巴细胞亚群（ CD4＋T细胞、CD8＋T细胞、NK细胞）含量。结果病例组外周血Th1细胞相关转录因子T-bet、RORγt、Foxp3及Th1细胞、Th1／Th2、TNF-α、IFN-γ含量较对照组显著降低,Th2细胞及IL-6、IL-10含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;病例组CD8＋3、CD8＋0、CD8＋6阳性的树突状细胞比例、CD4＋T细胞、NK细胞及补体C3、C4含量较对照组显著降低,CD8＋T细胞含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;TNF-α、IFN-γ含量与CD4＋T细胞、NK细胞、C3、C4含量呈正相关性（ r值分别为2．879、3．214、3．255和2．978, P ＜0．05）,与CD8＋T细胞含量呈负相关性（ r值分别为－3．146和－3．011, P ＜0．05）。 IL-6、IL-10含量与CD4＋T细胞、NK细胞、C3、C4含量呈负相关性（ r值分别为－2．124、－2．343、－3．423、－2．789、－2．993、－2．566、－3．758, P ＜0．05）,与CD8＋T细胞含量呈正相关性（ r值分别为3．465、3．129, P ＜0．05）。结论布鲁氏菌病患者外周血成熟树突状细胞数目减少,同时Th1／Th2细胞及相关细胞因子失衡,且与机体天然免疫和细胞免疫功能降低有关,这可能在布鲁氏菌病发生发展过程中发挥重要作用。     

Th1／Th2／Th17 T／reg 对鉴别儿童支气管哮喘与单纯性肺炎的意义

目的：检测Th1（IFN-γ＋）、调节T细胞（CD2＋5Foxp3＋）、辅助T 细胞17（Th17）及细胞因子IL-2、IL -4、IL-17和IL-21在儿童支气管哮喘以及肺炎患儿外周血中的表达,探讨其在儿童支气管哮喘发病中的作用以及与肺炎鉴别的意义。方法收集哮喘患儿32例,肺炎患儿37例作为研究对象,选取同期体检的22例健康儿童为对照组。分离外周血单个核细胞,采用流式细胞术检测CD 4＋IFN-γ＋、CD 4＋CD 2＋5Foxp 3＋、CD4＋Th17＋细胞百分率。采用酶联免疫吸咐（ ELISA）法检测3组IL-2、IL-4、IL-17、IL-21表达水平。结果哮喘组患儿外周血CD4＋CD2＋5 Foxp3＋细胞占CD4＋T细胞的比率明显低于肺炎组及对照组;哮喘组患儿者外周血CD4＋IFN-γ＋细胞比例明显低于肺炎组和对照组;哮喘组患儿者外周血CD4＋Th17＋细胞所占比例高于肺炎组及对照组,差异均有统计学意义（ P ＜0*．05）。哮喘组患儿外周血IL-2的浓度明显低于对照组（ P ＜00．5）,肺炎组患儿外周血IL-2浓度明显低于对照组（ P ＜0．05）,但与哮喘组比较差异无统计学意义（ P ＞0．05）。哮喘组患儿外周血 IL4-、IL-17和IL-21的浓度明显低于肺炎组和对照组,差异有统计学意义（ P ＜0．05）。结论 Th1／T2h ／Th17／Treg功能失调参与了儿童支气管哮喘的发病机制,临床上可以通过检测Th1／Th2／Th17／Treg相关细胞因子的变化评估儿童支气管哮喘病情并辅助与肺炎的鉴别。     

外源性LTB4对CIA小鼠Treg/Th17脾细胞分化的作用

目的探讨外源性白三烯B4(LTB4)对胶原诱导型关节炎(collagen-induced arthritis,CIA)小鼠脾细胞调节性T细胞(Treg)和Th17细胞分化的调节,进一步阐明LTB4在类风湿关节炎(RA)发病中的作用机制。方法建立CIA小鼠模型,取造模d28的脾细胞,体外实验分析外源性LTB4对Treg和Th17细胞分化的影响。应用流式细胞术检测CD4+CD25+Foxp3+细胞的数量,荧光定量PCR技术检测调控Treg和Th17细胞分化的特异性转录因子Foxp3和RORγt的mRNA的表达,酶联免疫吸附(ELISA)方法检测培养细胞上清IL-17的含量。结果成功建立CIA小鼠模型;造模d28分离小鼠脾细胞,体外培养加入鸡Ⅱ型胶原(CⅡ)共同孵育,随着LTB4浓度增加(0.01、0.1、1μmol·L-1),CD4+CD25+Foxp3+细胞数量相应减少,Foxp3 mRNA的表达相应降低;相反,IL-17的含量相应增加,RORγt mRNA的表达相应升高。结论LTB4抑制CIA模型脾细胞Treg细胞的分化,促进Th17细胞的分化,提示LTB4在CIA发病过程中具有一定的促炎活性。     

CD4+IL-10+调节性T细胞在特应性皮炎患者中的改变

摘要 目的 探讨特应性皮炎患者CD4+IL-10+调节性T细胞及其细胞因子的改变。方法 用流氏细胞仪直接免疫荧光法检测AD患者外周血CD4+IL-10+T细胞的绝对计数和百分率;用酶联免疫吸附试验检测外周血细胞因子IL-10、TGF-β的浓度。 结果AD患者CD4+IL-10+T细胞绝对计数为0.26+0.126×109/L,占CD4+T细胞的百分率是12.76+4.85%;而对照组CD4+IL-10+T细胞绝对计数为0.016+0.007×109/L,占CD4+T细胞的百分率是3.27+1.03%。经t检验,AD患者CD4+IL-10+T细胞绝对计数和百分率均高于对照组,有统计学差异（p <0.05）。 AD患者外周血IL-10浓度为126.59+59.46 pg/ml,高于正常对照组（31.47+10.96 pg/ml）,经t检验,有统计学差异（p<0.05）。AD患者外周血TGF-β浓度为407.23+155.88 pg/ml,高于正常对照组（359.47+99.21 pg/ml）,但经t检验,没有统计学差异（p>0.05）。CD4+IL-10+T细胞绝对计数、百分率均与外周血IL-10浓度呈正相关,有统计学意义（r=0.47,r=0.65, p <0.05）;而与外周血TGF-β无统计学相关性（r=0.15,r=0.21 , p >0.05）。结论 IL-10可能是Trl发挥功能的主要因子,通过下调由Thl和Th2细胞介导的炎症反应过程,参与免疫调节。     

重症急性胰腺炎病人外周血微RNA-143-3p、环氧合酶-2表达水平与辅助性T细胞17/调节性T细胞平衡的关系

目的 探究重症急性胰腺炎（SAP）病人血清微RNA-143-3p(miR-143-3p)、环氧合酶-2(COX-2)表达水平与辅助性T细胞17(Th17)/调节性T细胞（Treg）平衡的关系。方法 选取2017年12月至2020年9月榆林市第一医院诊治的103例急性胰腺炎（AP）病人进行研究，按APACHEⅡ评分将其分为SAP组（45例）和轻症急性胰腺炎（MAP）组（58例），另选取同期55例体检健康者为健康组。比较各组血清miR-143-3p、维甲酸相关孤核受体γt(RORγt) mRNA、COX-2 mRNA、叉头/翼状螺旋转录因子3(Foxp3) mRNA、Th17、Treg、Th17/Treg水平；分析SAP病人血清miR-143-3p、COX-2 mRNA表达水平与外周血Th17、Treg、Th17/Treg、RORγt mRNA、Foxp3 mRNA的相关性；分析SAP发生的影响因素。结果 MAP组、SAP组病人血清miR-143-3p(0.70±0.23、0.42±0.14比1.04±0.35)、Foxp3 mRNA表达水平（0.56±0.19、0.31±0.11比1.0...     

白介素-18对肝癌裸鼠皮下移植瘤浸润的CD4+T细胞子集的影响

目的探讨白介素-18(IL-18)影响肝癌裸鼠皮下移植瘤浸润的T细胞子集变化的机制。方法以HepG2细胞在裸鼠皮下接种,形成人肝癌皮下移植瘤的同时,以不同剂量IL-18分为4组治疗,4组分别腹腔注射IL-18 0.75,1.00,1.25,0(模型组)μg(0.1 mL)/只,治疗4周,另设正常对照组。观察肿瘤的生长速度和瘤体大小的变化。4周后处死取肿瘤组织和脾组织,免疫磁珠阴选组织中CD4+T细胞,流式细胞术检测各组肿瘤组织及脾组织中IL-17+/IFN-γ-CD4+T(Th17细胞)、IL-17-/IFN-γ+CD4+T(Th1)和IL-17+/IFN-γ+CD4+T细胞的变化。结果 HepG2细胞株建立了裸鼠肝癌皮下移植瘤模型,IL-18抑制人肝癌皮下移植瘤的形成及转移,治疗时间越长剂量越大,抑制效果越明显;与正常对照组比较,肿瘤组织中Th17和IL-17+/IFN-γ+CD4+T细胞浸润数目增加,Th1细胞降低,与脾组织中一致,与正常对照组比较有显著差异(P<0.05)。给IL-18后,随着剂量的增加,Th1细胞增加,IL-17+/IFN-γ+CD4+T细胞和Th17细胞浸润数目降低,给药组与模型组比较有显著差异(P<0.05或0.01)。结论 IL-17+/IFN-γ+CD4+T细胞数目在裸鼠移植瘤及其脾组织中增加,可能是在肿瘤环境中,促进其向Th17细胞分化;IL-18通过诱导IFN-γ的生成促进Th1细胞的生成,呈正反馈效应抑制肿瘤的增殖和转移。     

α-硫辛酸干预对自身免疫性甲状腺炎发病的影响

目的观察不同剂量硫辛酸干预对高碘诱发的自身免疫性甲状腺炎(autoimmune thyroiditis,AIT)的动物模型中CD4~+CD25~+调节性T细胞和Th17细胞的变化及影响,探讨硫辛酸治疗AIT的免疫学机制。方法选取饮用质量浓度50 mg·L-1碘化钠水的NOD.H-2h4雌鼠,注射不同剂量硫辛酸干预。采用HE染色法观察淋巴细胞浸润的情况并进行甲状腺炎症程度的评分;测定血清甲状腺球蛋白抗体(Tg Ab)的水平;再用免疫荧光染色流式细胞仪分析CD4~+CD25~+Foxp3~+调节性T细胞和Th17细胞比例变化;实时定量RT-PCR法检测Foxp3 mRNA、IL-17mRNA和RORγtmRNA的表达水平。结果与AIT非干预组相比,硫辛酸干预组NOD.H-2h4小鼠CD4~+CD25~+Foxp3~+调节性T细胞比例及Foxp3 mRNA表达量均明显升高(P<0.05);脾细胞中Th17细胞比例及其转录因子IL-17mRNA和RORγtmRNA表达水平均明显减低(P<0.01)。但不同剂量硫辛酸干预组之间比较无显著性差异(P>0.05)。同时,硫辛酸干预组NOD.H-2h4小鼠血清Tg Ab滴度以及甲状腺内淋巴细胞浸润的程度均较同周龄非干预组小鼠明显降低(P<0.05)。结论α-硫辛酸对AIT有治疗效果。     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

Dynamic Frequency of Blood CD4+CD25+ Regulatory T Cells in Rats with Collagen-induced Arthritis

CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the frequency of CD4+CD25+ Tregs in peripheral blood (PB) of collagen-induced arthritis (CIA) rats during the development of arthritis, to determine whether their frequency is involved in the immunoregulation of this disease. The results showed that normal rats had similar frequencies of CD4+CD25+ Tregs in PB during the experiment time, expressed as a percentage of CD4+CD25+Foxp3+ T cells among the CD4+ T lymphocyte population. In contrast, the frequency of CD4+CD25+Foxp3+ T cells in CIA rats was found to change during the development of arthritis. In CIA rats, there is a significant negative correlation between the frequency of CD4+CD25+Foxp3+ T cells and paw swelling (r=-0.786, p< 0.01). The relationship between the frequency of CD4+CD25+Foxp3+ T and immune activation was not found in normal rats. During the time course, the frequency of CD4+CD25+Foxp3+ T was lower in CIA rats than in normal ones. The data suggest that the frequency of PB CD4+CD25+ Tregs may be a promising marker for arthritis activity.     

CD4+ Foxp3+ regulatory T cells induced by TGF-β, IL-2 and all-trans retinoic acid attenuate obliterative bronchiolitis in rat trachea transplantation

Obliterative bronchiolitis (OB) is the major obstacle for long-term allograft survival in lung transplantation, and the underlying mechanism is still not well understood. Regulatory T cells (Tregs) have been shown to be essential in the maintenance of immune tolerance. In this study we investigated the role of Tregs in protecting OB in rat. We show that the combination of TGF-β, Interleukin (IL)-2, and all-trans retinoic acid (atRA) could induce naïve rat CD4⁺CD25⁻ T cells to differentiate into CD4⁺CD25⁺Foxp3⁺ T cells in vitro, and they acquired suppressive function. In a rat orthotopic tracheal transplantation OB model, the adoptive transfer of the induced Tregs reduced symptoms of airway obliteration and fibrication of grafts when compared with adoptive transfer of control cells without suppressive property. Moreover, recipients treated with the induced Tregs secreted high level of immunosuppressive cytokine TGF-β and IL-10, and low level of pro-inflammatory cytokines IL-17, IFN-γ, IL-6, and MCP-1, and had fewer effector T cells including Th17 cells and Th1 cells in the graft. Taken together, these findings suggest that in vitro induced Tregs by the combination of TGF-β, IL-2, and atRA are effective in protecting rat trachea allograft rejection through the inhibition of effector T cells and their function. These datas implicate new therapies to prevent OB and allograft rejection in human lung transplantation.     

Rapamycin inhibits differentiation of Th17 cells and promotes generation of FoxP3+ T regulatory cells

Reciprocal differentiation of immunosuppressive CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) and proinflammatory IL-17-producing cells (Th17) from naïve CD4 cells is contingent upon the cytokine environment. Using MACS-purified CD4 cells, we found that rapamycin and cyclosporine A (CsA) potently inhibited the TGFβ and IL-6-induced generation of IL-17-producing cells. Intriguingly, rapamycin promoted, while CsA markedly inhibited, TGFβ-mediated generation of Tregs. The aforementioned effects of rapamycin and CsA were also observed for Flow-sorted CD4⁺CD25⁻ T cells, indicating that the effect of these two immunosuppressive agents was based on their action on de novo generation of Tregs and Th17 cells from naïve CD4 cells. Our observation suggests a distinct mode of immunosuppressive action and tolerance induction by rapamycin and CsA. The capacity of rapamycin to generate immunosuppressive Tregs and to suppress differentiation of pathogenic Th17 cells furthers our understanding of the basis for the therapeutic immunosuppressive effects of rapamycin in patients with autoimmune diseases and allo-transplantation reactions.