再生障碍性贫血患者外周血T-bet和GATA-3转录因子基因表达失衡的分析

目的探讨再生障碍性贫血(aplastic anemia,AA)患者外周血单个核细胞中T-bet/GATA-3的比率与Th1/Th2细胞失衡的关系。方法采用RT-PCR法检测30例初诊AA患者及20名健康人外周血单个核细胞(PBMC)转录因子T-bet、GATA-3 mRNA的表达水平,ELISA法检测血浆IFN-γ和IL-4水平。结果 AA组PBMC中T-bet mRNA及血浆IFN-γ水平均显著高于正常对照组(P分别<0.01),且T-bet/GATA-3 mRNA的比率较对照组明显升高(P<0.01),Th1类细胞因子IFN-γ的水平与T-bet/GATA-3的比率成正相关(r=0.84),而Th2类细胞因子IL-4的水平降低,与T-bet/GATA-3的比率成负相关(r=0.75)。结论 T-bet和IFN-γ表达的增高在AA的发病中可能起到重要作用;AA患者T-bet/GATA-3的比率升高可作为评价Th1/Th2细胞失衡的另一重要指标,在协助临床对AA诊断中起重要作用。     

再生障碍性贫血患者外周血T-bet和GATA-3转录因子基因表达失衡的分析

目的探讨再生障碍性贫血（aplastic anemia,AA）患者外周血单个核细胞中T-bet／GATA-3的比率与Thl／Th2细胞失衡的关系。方法采用RT—PCR法检测30例初诊AA患者及20名健康人外周血单个核细胞（PBMC）转录因子T-bet、GATA-3mPR NA的表达水平,ELISA法检测血浆IFN—Y和IL-4水平。结果从组PBMC中T-betmRNA及血浆IFN-Y水平均显著高于正常对照组（P分别〈0．01）,且T-bet／GATA-3m RNA的比率较对照组明显升高（P〈0．01）,Thl类细胞因子IFN-7的水平与T-bet／GATA-3的比率成正相关（r=0．84）,而Th2类细胞因子IL-4的水平降低,与T-bet／GATA-3的比率成负相关（r=0．75）。结论T-bet和IFN-7表达的增高在AA的发病中可能起到重要作用;从患者T-bet／GATA-3的比率升高可作为评价Thl／Th2细胞失衡的另一重要指标,在协助临床对AA诊断中起重要作用。     

转录因子T-bet/GATA3在EV71感染患儿外周血单个核细胞中的表达及其与相关细胞因子的关系

目的探讨转录因子T-bet/GATA3在肠道病毒71型(enterovirus 71,EV71)感染患儿外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的表达及其与相关细胞因子的关系。方法收集42例EV71感染患者外周血标本,其中急性期28例、恢复期14例,另外健康对照组21例。采用实时荧光定量PCR(QRT-PCR)检测PBMC中T-bet和GATA3 mRNA水平;酶联免疫吸附(ELISA)法检测血浆IFN-γ和IL-4的蛋白浓度。结果急性EV71感染患者PBMC中的T-bet mRNA表达和血浆IFN-γ水平明显低于健康对照组(P<0.05),急性EV71感染患者GATA3 mRNA的表达和IL-4水平则明显高于健康对照组(P<0.05)。结论急性EV71感染患者存在Th2优势分化,可能与转录因子T-bet表达下调、GATA3表达上调有关。监测EV71感染患者外周血Th1/Th2细胞特异性转录因子与相关细胞因子的水平,有助于对病情的判断。     

转录因子GATA-3与T-bet对婴幼儿毛细支气管炎的免疫调节

目的:探讨转录因子GATA-3与T-bet对婴幼儿毛细支气管炎(毛支炎)的免疫调节作用。方法 :对30例毛支炎(毛支炎组)和30例正常儿童(对照组)抽取抗凝血2 mL和促凝血2 mL,2 mL抗凝血以TIANGEN总RNA提取试剂盒提取抗凝血总RNA,用逆转录聚合酶链式反应(RT-PCR)检测细胞GATA-3,T-bet的mRNA表达水平。2 mL促凝血3 000r/min高速离心,取血清500μL,采用酶联免疫吸附法(ELISA)检测血清中干扰素-γ(IFN-γ)和白细胞介素-4(IL-4)水平。结果:毛支炎组血清IL-4水平〔(22.0±4.89)pg/ml〕明显高于对照组〔(13.57±5.14)pg/ml〕(P<0.05),而IFN-γ水平〔(6.89±1.79)pg/ml〕低于对照组〔(10.99±2.59)pg/ml〕(P<0.05)。毛支炎组和对照组GATA-3 mRNA表达水平分别为(14.49±3.75)和(11.02±2.11),T-bet mRNA分别为(5.87±1.29)和(10.99±2.59)。显示毛支炎组GATA-3mRNA表达高于对照组(P<0.05),而T-bet mRNA表达较对照组减少(P<0.05)。毛支炎组IFN-γ浓度与T-bet mRNA表达呈正相关(r=0.77,P<0.05)。IL-4与GATA-3 mRNA表达呈正相关(r=0.95,P<0.05)。IFN-γ与IL-4水平呈负相关(r=-0.61,P<0.05)。结论:①毛支炎组患儿血清IL-4水平增高,IFN-γ降低,存在以Th2细胞过度分化为特征的T辅助细胞分化失衡。②毛支炎组患儿T细胞失衡受到转录因子GATA-3与T-bet调控,与毛支炎患儿Th1/Th2失衡密切相关,可作为衡量Th1/Th2平衡之新指标。     

丹参协同地塞米松对哮喘大鼠T-bet/GATA-3及Th失衡的调控研究

目的探讨丹参协同地塞米松对哮喘大鼠T-bet/GATA-3及Th失衡的调控作用。方法随机将80只SD大鼠分为哮喘模型组;正常对照组;丹参治疗组;联合用药组（丹参协同地塞米松治疗）,每组20只。测定气道反应性;肺组织苏木素伊红（HE）染色观察病理改变;蛋白质印迹法（western-blots）检测T-bet和GATA-3在肺组织中的表达;酶联免疫吸附试验（ELISA）检测肺组织匀浆白细胞介素4（IL-4）和白细胞介素5（IL-5）水平。结果丹参组和联合用药组大鼠肺部病理学切片呈现肺部炎症改变较哮喘组减轻,以联合用药组减轻更明显。大鼠气道阻力示哮喘组>丹参组>联合用药组>对照组(P 均< 0.05),且各组随着组胺浓度的升高气道阻力增高明显。蛋白质印迹法检测肺组织中T-bet水平对照组>联合用药组>丹参组>哮喘组(P 均< 0.01),而GATA-3水平则与之相反。肺组织IFN-γ蛋白水平对照组>联合用药组>丹参组>哮喘组(P 均< 0.01),而IL-4水平则与之相反。肺T-bet/GATA-3水平与IFN-γ蛋白水平呈正相关(r=0.787,P < 0.01) ,与IL-4 蛋白水平呈负相关( r=-0.816,P < 0.01) ,与IFN-γ/IL-4比值呈正相关(r=0.893,P < 0.01)。结论丹参注射液可上调T-bet/GATA-3水平,进而纠正Th1/Th2失衡,与地塞米松有协同发挥抗气道炎症作用。     

复合微量营养素对C57BL糖尿病小鼠Th1、Th2型细胞因子表达的调控作用

目的通过研究复合微量营养素(MC)对C57BL糖尿病小鼠Th1、Th2型细胞因子抗原表达的影响,探讨AM拮抗糖尿病的分子作用机制。方法应用小剂量多次注射STZ的方法(multiplelowdoseofstreptozotocin,MLDS)制备小鼠IDDM模型,经灌胃联合投予硒(Se)、维生素E(VE)、钒(V)、铬(Cr),应用流式细胞术观察表达Th1型细胞因子(TNFα、IFNγ)和Th2型细胞因子(IL4、IL10)抗原的淋巴细胞数百分比。结果IDDM小鼠外周血表达TNFα、IFNγ抗原的淋巴细胞数百分比明显升高(P<001),而外周血、脾组织表达IL10抗原的淋巴细胞数百分比明显降低(P<001);应用MC可显著降低IDDM小鼠外周血淋巴细胞TNFα的表达(P<001),显著升高外周血淋巴细胞IL10、脾淋巴细胞IL4的表达(P<001,P<005)。结论MC可通过下调Th1型细胞因子、上调Th2型细胞因子的基因表达,来调节IDDM时的免疫失衡,减轻胰岛自身免疫性损害,拮抗糖尿病的发生发展。     

过敏性哮喘患儿外周血单个核细胞mir-29c与Thl/Th2细胞平衡的相关性分析

目的 分析过敏性哮喘(AA)患儿外周血单核细胞微小RNA 29c(mir-29c)表达及与CD4+辅助性T细胞1/CD4+辅助性T细胞2(Th1/Th2)平衡的相关性。方法 将2015年10月-2018年12月本院收治的94例AA患儿分为稳定组32例,轻度急性发作组27例,中度急性发作组35例;另取健康体检儿童40例为对照组。采集所有受试者儿童外周血,分离血清和单个核细胞(PBMC),检测mir-29c、Th1/Th2、IFN-γ、IL-4、T盒子转录因子21(T-bet)、GATA结合蛋白3(GATA3)蛋白水平。结果 与对照组比较,稳定组、轻、中度急性发作组AA患儿PBMC中mir-29c水平、Th2亚群比例、GATA3蛋白水平及血清IL-4水平依次升高,PBMC中Th1亚群比例、Th1/Th2、T-bet蛋白水平、T-bet/GATA3及血清IFN-γ水平、IFN-γ/IL-4依次降低,两两比较差异均有统计学意义(P＜0.05)。AA患儿外周血mir-29c水平与Th1/Th2、IFN-γ/IL-4、T-bet/GATA3均呈负相关(r=-0.823、-0.789、-0.804,P＜0.05)。结论 mir-29c在AA患儿外周血单个核细胞中上调表达,可能通过Th1/Th2平衡影响AA发生。     

T-bet / GATA3转录因子与5-HTT基因多态性对哮喘发病的影响

目的 探讨T-bet / GATA3转录因子与5-HTT基因多态性在哮喘发病中的作用。方法 收集221例壮族哮喘患者和208例壮族健康对照者,采用PCR重测序法进行5-HTT基因rs3794808位点基因分型,RT-PCR检测T-bet和GATA3 mRNA相对表达量,ELISA测定血清中IL-2和IL-5浓度。结果 病例组CC基因型和C等位基因频率均高于对照组（x2=7.137,P=0.027;x2=7.265,P=0.007）,CC基因型是哮喘的危险因素（OR=3.090,95%CI:1.804~8.810）;与对照组相比,病例组IL-2浓度低而IL-5浓度高（t=8.969、119.908,P均小于0.001）;CC基因型哮喘患者T-bet mRNA表达量和IL-2浓度低于TT基因型（F=4.736,P =0.009;F=7.512,P =0.001）,而GATA3 mRNA和IL-5浓度高于TT基因型（F=19.947、40.234,P均小于0.001）;哮喘患者T-bet mRNA与IL-2浓度呈正相关（r=0.335,P <0.001）,与IL-5浓度呈负相关（r=-0.221,P =0.001）,GATA3 mRNA与IL-2浓度呈负相关（r=-0.289,P<0.001）,与IL-5浓度呈正相关（r=0.608,P<0.001）,T-bet/GATA3比值与IL-2浓度呈正相关（r=0.402,P <0.001）,与IL-5浓度呈负相关（r=-0.472,P <0.001）。结论 5-HTT基因的多态性可作为遗传易感背景影响T-bet / GATA3转录因子的表达,从而造成Th1/Th2失衡来参与哮喘的发病过程。这或许能成为哮喘治疗的途径之一。     

Cytokine mRNAs in the nasal-associated lymphoid tissue during influenza virus infection and nasal vaccination

Intranasal immunization with a current inactivated influenza vaccine together with an adjuvant (cholera toxin B subunit supplemented with a trace amount of whole toxin, CTB*) was confirmed in BALB/c mice to mimic influenza virus (A/PR/8/34, H1N1) infection with respect to mucosal IgA antibody responses, in which IgA antibody-forming cell responses in the nasal-associated lymphoid tissue (NALT) were involved with a peak around 7 days after infection or vaccination. Next, the expression of various cytokine mRNAs in the NALT was compared in mice either infected with viruses or immunized with CTB*-combined vaccine, to examine Th cell and cytokine regulation of mucosal IgA antibody responses. In infected mice, strong IL-2, weak IL-4, strong IL-6 and strong IFN-gamma mRNA expressions were induced during early days of infection; especially, IFN-gamma mRNA was expressed by both CD4(+) and CD8(+) T cells around 7 days after infection. In mice given CTB*-combined vaccine, weak IL-2, strong IL-4, strong IL-6 and weak IFN-gamma mRNA expressions were induced during early days of vaccination; especially, IL-4 mRNA was expressed by CD4(+) T cells. Thus, IL-6 mRNAs were expressed strongly in both infected and vaccinated mice. The IFN-gamma-rich cytokine mRNA profiles in the infected mice were reflected upon serum IgG2a-rich Ab responses, while the IL-4-rich profiles in the vaccinated mice were reflected upon the IgG1-rich Ab responses. Thus, influenza virus infection and CTB*-combined nasal vaccine induced Th1 dominant and Th2 dominant cytokine profiles, respectively, while the similarity of mucosal IgA antibody responses between infection and vaccination could be explained by the appearance of IL-6 mRNAs.     

Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses

Induction of peripheral tolerance can be facilitated when the antigen is linked to the B subunit of cholera toxin (CTB), an efficient mucosal carrier. In the present study, a genetic fusion molecule of Bet v 1 and CTB was produced to test whether mucosal application of this construct would lead to suppression of Th2 responses. Intranasal pretreatment of BALB/c mice with rCTB-Bet v 1 prior to allergic sensitisation with the allergen significantly decreased IgE but markedly increased allergen-specific IgG2a levels in sera as well as IFN-gamma production of splenocytes. This Th1 shift was supported by an increased T-bet/GATA3 mRNA ratio. IL-5 production within the airways was suppressed after the pretreatment with rCTB-Bet v 1, while local allergen-specific IgA antibodies were markedly enhanced by pretreatment with the construct. Upregulation of Foxp3, IL-10 and TGF-beta mRNA expression was detected in splenocytes after pretreatment with unconjugated allergen but not with the fusion molecule, indicating that antigen conjugation to a mucosal carrier modifies the immunomodulating properties of an antigen/allergen.     

Intranasal immunization with a replication-deficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity

A vaccine vector, designated AdV-gH, was constructed by inserting the complete open reading frame of MCMV gH under control of the human CMV IE-1 promoter into the E-1 region of a replication-deficient adenovirus 5. In vitro infection of QB1-293 cells and mouse embryo cells with AdV-gH resulted in expression of MCMV gH detected by IFA. Immunization of BALB/c mice with AdV-gH (1 x 10(7) PFU) given by the intranasal route induced a humoral response with antibody detected in serum of 100% of vaccines. Antibody to MCMV gH was also detected in the bronchoalveolar lavage, fecal suspensions and vaginal washings. The viral titer of lung and salivary gland of immunized mice 10 days after intranasal challenge with MCMV (1 x 10(5) PFU) were significantly reduced compared to controls, but virus infection was not prevented. Re-exposure of mice to AdV-gH 30 days after primary immunization induced a significant boost of serum antibody response. When rechallenged with MCMV intranasally, these mice had further reduction of MCMV titers in the lung and salivary glands. Such a strategy may be important in reducing horizontal transmission of CMV infections across mucosal surfaces and in altering host immunity to CMV.     

Lung pathology and immediate hypersensitivity in a mouse model after vaccination with pertussis vaccines and challenge with Bordetella pertussis

While evaluating vaccine efficacy against clinical Bordetella pertussis isolates in mice, after challenge vaccinated mice showed increased lung pathology with eosinophilia, compared to challenged, non-vaccinated animals. This led us to study bacterial clearance, lung pathology, lung TNF-alpha expression, and parameters of immediate hypersensitivity (IH), being serum IgE levels, eosinophil numbers in the bronchoalveolar lavage fluid, and ex vivo IL-4, IL-5, IL-10, IL-13, and IFN-gamma production by the bronchial lymph node cells. BALB/c mice received a combined Diphtheria (D), Tetanus (T), Poliomyelitis, and whole-cell Pertussis vaccine (WCV), a combined D, T, and three-component acellular Pertussis vaccine (ACV), aluminium hydroxide adjuvant, or PBS, 28 and 14 days before B. pertussis infection. Similarly treated non-infected mice were taken as a control. Infection induced pathology; this induction was stronger after (especially WCV) vaccination. WCV but not ACV vaccination induced TNF-alpha expression after challenge. After challenge, IH parameters were strongly increased by (especially ACV) vaccination. Vaccinated IL-4 KO mice showed similar clearance and pathology, in the absence of IgE and with reduced numbers of eosinophils. Vaccinated (Th1-deficient) T-bet KO mice showed reduced clearance and similar pathology. In summary, after challenge vaccination increased lung pathology, TNF-alpha expression (only WCV), and IH parameters. Th1 cells were critical for clearance.     

转录因子GATA-3对呼吸道合胞病毒感染大鼠Th2型细胞因子表达的调控研究

目的 探讨呼吸道合胞病毒（RSV）感染时肺GATA-3表达对Th2类细胞因子分泌的调控作用.方法 将20只SD大鼠按数字表法分为对照组、RSV感染组（RSV滴鼻法建立RSV感染模型）,每组10只.在第7周测定气道反应性;取肺组织苏木素伊红（HE）染色观察病理改变;通过蛋白质印迹法（western-blots）检测GATA-3在肺组织中的表达;酶联免疫吸附试验（ELISA）检测肺组织匀浆白细胞介素4（IL-4）和白细胞介素5（IL-5）水平.结果 RSV感染组大鼠肺部病理学切片呈现典型的间质性肺炎表现,而且RSV感染组气道阻力增高程度明显高于对照组大鼠.RSV感染组肺组织中GATA-3/GAPDH相对灰度值（1.743±0.21）高于对照组（1.231±0.31）（t=15.43,P＜0.01）.RSV感染组IL-4[（142.3±12.3）ng/L]及IL-5蛋白水平[（167.5±14.2）ng/L]均高于对照组[（69.2±11.6）ng/L,（86.6±14.3）ng/L]（P＜0.01）.肺GATA-3蛋白水平与IL-4蛋白及IL-5蛋白水平呈正相关（r=0.864,0.756,P＜0.01）.结论 RSV感染可以上调GATA-3在肺组织中的表达,并与Th2型炎症反应呈正相关.     

Vaccination with DNA encoding ORFF antigen confers protective immunity in mice infected with Leishmania donovani

The gene ORFF is part of the multigenic LD1 locus on chromosome 35 that is frequently amplified in Leishmania. The function of ORFF is unknown. The gene encoding ORFF was cloned into a eukaryotic expression vector downstream to the cytomegalovirus (CMV) promoter. BALB/c mice were injected intramuscularly with ORFF DNA and challenged with Leishmania donovani promastigotes. Vaccination with ORFF gene induced both humoral and cellular immune response against ORFF, which provided significant level of protection against challenge with L. donovani. A qualitative PCR was used to determine whether activation of Th1 cells develops selectively in response to this ORFF DNA vaccine. The results indicated that mRNA for IFN-gamma was significantly induced in immunized mice. No significant change in IL-4 mRNA expression was observed in mice immunized with ORFF DNA vaccine versus mice immunized with control plasmid. Thus, DNA immunization may offer an attractive alternative strategy against leishmaniasis.     

白细胞介素27在抗病毒免疫中的作用及临床应用前景

IL-27是IL-6/IL-12家族细胞因子,可发挥广泛的免疫调节作用.IL-27可通过STAT1依赖或者非依赖途径抑制GATA-3(Th2分化的关键转录因子),从而有利于Th1的分化.IL-27引起naive T细胞产生细胞间黏附分子1,以STAT1依赖方式有利于Th1的分化.IL-27体外还可通过诱导STAT1抑制...     

Human schistosomiasis mansoni: intensity of infection differentially affects the production of interleukin-10, interferon-gamma and interleukin-13 by soluble egg antigen or adult worm antigen stimulated cultures

The effect of the intensity of infection (eggs per gram faeces, epg) on the production of interferon-gamma (INF-gamma), interleukin (IL)-10 and IL-13 by peripheral blood mononuclear cells (PBMCs) from individuals living in a Schistosoma mansoni-endemic area was evaluated. In vitro stimulation of PBMCs with soluble egg antigen (SEA) resulted in significantly higher secretion levels of IFN-gamma in egg-negative individuals compared with those with an intensity of infection of more than 100 epg. In contrast, the egg-positive group produced significantly higher amounts of IL-10. Levels of IL-13 did not differ significantly between egg-positive and egg-negative groups. These findings suggest that IL-10 is an important cytokine in the control of the T helper cell (Th) type 1 responses during human S. mansoni infection, shifting the immune response from Th0 in egg-negative individuals from an endemic area to a Th2 polarization in chronic infected individuals.