负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响

目的探讨负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响。方法将CTLA4Ig重组腺病毒与Wistar大鼠未成熟树突状细胞于37℃共孵育6h后,经尾静脉注射该大鼠作实验组,另外分别设立Wistar大鼠未成熟树突状细胞、CTLA4Ig重组腺病毒、生理盐水经尾静脉注射为对照。1周后,用0.3%戊巴比妥麻醉各组大鼠后抽血检测CTLA4Ig。取脾脏,经流式细胞术分选出Th1、Th2细胞及CD4 T细胞,行混合淋巴细胞培养检测Th细胞的增殖。用免疫组织化学法检测Th1、Th2细胞的比例。结果实验组血清CTLA4Ig水平(0.654±0.13)显著高于CTLA4Ig重组腺病毒组(0.392±0.10,P<0.01),树突状细胞组及生理盐水组未检出。实验组Th1细胞的增殖指数(742±161)、Th1/Th2(0.16±0.05)均显著低于各对照组(分别与未成熟树突状细胞组、CTLA4Ig重组腺病毒组、生理盐水组相比,P均<0.01);而Th2细胞的增殖指数(9162±598)显著高于各对照组(P均<0.01)。结论负载CTLA4Ig重组腺病毒的未成熟树突状细胞可显著抑制大鼠Th1细胞增殖,促进Th2细胞增殖,使Th细胞由Th1向Th2显著偏移,诱导有效的免疫耐受。     

慢性乙肝患者树突状细胞对Th1/Th2分化的影响

探讨慢性乙肝患者树突状细胞(dendritic cells,DC)对CD4+Th细胞亚群分化的影响。分离慢性乙肝患者外周血单个核细胞(PBMC),以rhIL-4(50 ng/ml)、rhGM-CSF(10 ng/ml)和rhTNF-α(100 u/ml)诱导培养DC。以流式细胞仪检测DC表面CD1a、CD83、CD80、CD86、HLA-DR分子表达情况。MTT法检测DC刺激同种异体淋巴细胞增殖能力。免疫磁珠分离外周血CD4+T细胞亚群,PMA+Ionomycin刺激后胞内荧光染色,流式细胞仪检测辅助性T细胞(helper T cell,Th)内特征性细胞因子IFN-γ/IL-4以判断Th1/Th2分化。ELISA法检测DC或Th细胞培养上清中IL-6、IL-12、IFN-γ和IL-4的含量。结果:慢性乙肝患者的DC表达CD1a、CD83、CD80、CD86、HLA-DR分子水平明显低于正常人(P<0.01);培养至第7天,慢性乙肝患者DC分泌的IL-12水平低于正常人(P<0.01),而分泌的IL-6水平增高(P<0.05)。与正常人相比,慢性乙肝患者外周血中Th1细胞占CD4+T细胞的百分比较低(P<0.01),其Th细胞培养上清中IFN-γ的量也较低(P<0.01)。患者DC与同种异体的健康人Th细胞共培养,刺激Th1型细胞因子IFN-γ产生的能力低于正常人(P<0.01)。慢性乙肝患者体内DC功能的异常可能导致了外周血Th1细胞分化不足。     

负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响

目的 探讨负载CTLA4Ig重组腺病毒的未成熟树突状细胞对大鼠Th细胞增殖的影响.方法 将CTLA4Ig重组腺病毒与Wistar大鼠未成熟树突状细胞于37C共孵育6h后,经尾静脉注射该大鼠作实验组,另外分别设立Wistar大鼠未成熟树突状细胞、CTLA4Ig重组腺病毒、生理盐水经尾静脉注射为对照.1周后,用0.3%戊巴比妥麻醉各组大鼠后抽血检测CTLA4Ig.取脾脏,经流式细胞术分选出Th1、Th2细胞及CD4+T细胞,行混合淋巴细胞培养检测Th细胞的增殖.用免疫组织化学法检测Th1、Th2细胞的比例.结果 实验组血清CTLA4Ig水平(0.654±0.13)显著高于CTLA4Ig重组腺病毒组(0.392±0.10,P＜0.01),树突状细胞组及生理盐水组未检出.实验组Th1细胞的增殖指数(742±161)、Th1/Th2(0.16±0.05)均显著低于各对照组(分别与未成熟树突状细胞组、CTLA4Ig重组腺病毒组、生理盐水组相比,P均＜0.01);而Th2细胞的增殖指数(9162 ±598)显著高于各对照组(P均＜0.01).结论 负载CTLA4Ig重组腺病毒的未成熟树突状细胞可显著抑制大鼠Th1细胞增殖,促进Th2细胞增殖,使Th细胞由Th1向Th2显著偏移,诱导有效的免疫耐受.     

CTLA4-Ig在树突状细胞中的表达及效应研究

目的 获得CTLA4-Ig转基因修饰的DC细胞,分析表达产物CTLA4-Ig对T细胞活化功能的影响。方法 采用脂质体转染方法将CTLA4-Ig表达载体转入DC细胞,通过ELISA法鉴定转染DC细胞48和72h培养上清液及稳定表达的培养上清液,分离Balb/c小鼠和C57BL/10J小鼠淋巴细胞为反应细胞和刺激细胞,观察CTLA4-Ig对同种细胞混合培养反应的影响。结果 将鼠CTLA4-Ig表达载体转入DC细胞,获得瞬时表达及稳定表达,CTLA4-Ig可抑制单向混合淋巴细胞反应,且效应亦呈剂量依赖性。结论 鼠CTLA4-Ig表达产物对同种细胞刺激的增殖反应有抑制作用,其抑制作用随表达载体转染细胞培养上清液的增加而增强。同时获得了稳定表达CTLA4-Ig融合蛋白的CTLA4-Ig转基因修饰的DC细胞,为以后的工作奠定了基础。     

CTLA4Ig修饰树突状细胞对实验动物淋巴细胞增殖及胞毒效应的影响

目的：探讨CTLA4Ig修饰的DC对实验动物免疫功能的影响。方法：将经CTLA4Ig基因修饰或未修饰Dc腹腔注射C57BL／6致敏小鼠，以致敏或未致敏C57BL／6单个核细胞作为反应细胞，以未修饰DC细胞及修饰DC为刺激细胞，共培养6天，采用MTT比色法检测细胞增殖，乳酸脱氢酶法测定细胞毒活性。结果：CTLA4Ig融合蛋白对未修饰DC致敏或未致敏小鼠的同种细胞刺激的增殖反应有明显的抑制作用。CTLA4Ig修饰DC诱导不同组小鼠淋巴细胞增殖反应均明显降低，CTLA4Ig融合蛋白对CTL细胞毒活性有显著抑制作用。CTLA4Ig修饰DC对不同组小鼠CTL细胞毒活性均具有抵抗作用，未修饰DC细胞对未致敏小鼠以及未修饰DC对致敏小鼠CTL细胞毒活性敏感。结论：稳定表达CTLA4Ig融合蛋白的DC诱导显著降低同种小鼠淋巴细胞的增殖反应和对CTL细胞毒活性的抵抗。     

CTLA4Ig修饰的树突状细胞在体外对淋巴细胞增殖及胞毒效应的影响

目的:探讨CTLA4Ig修饰的树突状细胞(DC)对T细胞增殖和细胞毒性T细胞(CTL)细胞毒活性的影响。方法:采用脂质体转染法,将质粒pG/CTLA4Ig转入DC。采用ELISA和SDS-PAGE鉴定转染质粒pG/CTLA4Ig的DC培养上清。以C57BL/6小鼠的单个核细胞作为反应细胞,以未修饰的DC及修饰的DC作为刺激细胞,共培养6 d,用MTT比色法检测细胞的增殖。用乳酸脱氢酶法和ELISA法,分别测定细胞毒活性及T细胞凋亡。结果:CTLA4Ig融合蛋白和修饰的DC,对同种细胞刺激的增殖反应有明显地抑制作用;而未修饰的DC可显著诱导淋巴细胞增殖反应。CTLA4Ig融合蛋白和修饰的DC,对特异性CTL的细胞毒活性有明显地抑制作用,且可诱导T细胞凋亡。结论:稳定表达CTLA4Ig融合蛋白的DC,对T细胞增殖和对CTL的细胞毒活性具有明显地抑制作用,并可诱导T细胞凋亡。     

树突状细胞和Th细胞分化的关系

树突状细胞 (dendriticcells ,DC )根据其来源、表型及分泌的细胞因子不同可分为髓系DC (DC1)和淋巴系DC (DC2 )。DC1诱导Th1细胞应答 ,DC2诱导Th2细胞应答。本文就DC和Th细胞分化的关系作一简要综述 ,包括DC分类及其对Th细胞分化的诱导以及Th细胞亚类对DC的反馈作用     

乳杆菌对原代淋巴细胞中Th1/Th2细胞平衡的影响

目的:分析7种乳杆菌对原代淋巴细胞增殖和细胞因子(CK)分泌的作用,进而探讨其对Th1/Th2细胞平衡的影响。方法:用不同种属、不同浓度的活的/热致死的乳杆菌体外作用于小鼠脾淋巴细胞培养60 h后,采用MTT比色法检测淋巴细胞的增殖效果。用ELISA法检测Th1型细胞因子(IL-12、IFN-γ)、Th2型细胞因子(IL-4、IL-10)和调节型细胞因子(TGF-β)的分泌量。结果:活的/热致死的乳杆菌单独作用,就能促进淋巴细胞体外增殖并表现出剂量依赖关系(P<0.05)。当菌的浓度为107集落形成单位(CFU)/mL(即细菌与细胞的比例为10∶1)时,热致死的发酵乳杆菌和嗜酸乳杆菌的免疫活性近似于活菌。而且,这两株热致死菌还可适当提高淋巴细胞分泌IL-12和IFN-γ,抑制IL-4、IL-10和TGF-β的分泌,使其IFN-γ/IL-4的比值(代表Th1/Th2细胞平衡)均显著高于刀豆蛋白A(ConA)对照组(P<0.05)。结论:乳杆菌可通过提高淋巴细胞的IFN-γ/IL-4分泌率来促进Th1优势状态的Th1/Th2细胞平衡,并具有菌株特异性。     

腹腔镜子宫肌瘤剔除术对Th1/Th2细胞平衡的影响

目的:为了探讨腹腔镜子宫肌瘤剔除术对Th1/Th2细胞平衡的影响。方法:择期子宫肌瘤患者4 0例,分别采用腹腔镜手术和常规开腹手术,测定术前、术后2、2 4、4 8小时Th1、Th2细胞数量及血清IL 18、IL 10水平。结果:两组术后2小时均出现Th1/Th2细胞平衡向Th2反应转换,Th1细胞、Th1/Th2比值、IL 18下降(腹腔镜组:P <0 0 5 ,P <0 0 1,P <0 0 5 ;开腹组:P <0 0 1,P <0 0 1,P <0 0 1) ,而Th2细胞、抗炎因子IL 10上升(腹腔镜组:P <0 0 1,P <0 0 1;开腹组:P <0 0 1,P <0 0 1)。但腹腔镜组术后2 4小时各项指标即恢复,而开腹组各项指标的变化与术后2小时类似,并持续至术后4 8小时。结论:腹腔镜子宫肌瘤剔除术对Th1/Th2细胞平衡影响小,恢复快,优于开腹手术。     

子痫前期患者外周血树突状细胞对Th1/Th17细胞分化的影响

目的 观察正常妊娠妇女和子痫前期患者外周血单个核细胞(PBMC)来源的树突状细胞(DC)对Th1、Th17细胞分化的影响.方法 实验组为子痫前期疾病患者32例;对照组为正常妊娠妇女20例.分离正常妊娠组和子痫前期组PBMC,经贴壁获得单核细胞,加GM-CSF,IL-4和LPS培养诱导为成熟DC,流式细胞术检测表面分子标志CD14,CD80,CD83,CD86的表达,ELISA检测培养上清液中IL-23的含量.磁珠分选出CD4+T淋巴细胞,与正常妊娠孕妇外周血单核细胞来源的树突状细胞(N-DC)共同培养,同时添加细胞因子IL-2,或与子痫前期患者外周血单核细胞来源的树突状细胞(P-DC)、IL-2共同培养;或与N-DC共同培养,同时添加细胞因子IL-1p、IL-6;或与P-DC共同培养,并添加细胞因子IL-1β、IL-6.以上各组培养到第6天用流式细胞术检测CD4+ IFN-γ+ Th1、CD4+ IL-17+ Th17细胞比例.结果 P-DC中CD83、CD80、CD86的表达高于N-DC,差异有统计学意义(P＜0.05).P-DC与不同的细胞因子共同作用,促使CD4+T细胞分化为Th1、Th17细胞的能力高于N-DC,差异有统计学意义(P＜0.01).结论 子痫前期患者外周血DC表型和功能的改变可能与患者免疫失衡有一定关系.     

CTLA4—Ig影响T细胞功能及机理的初步研究

目的：研究CTLA4-Ig融合蛋白对外周血T淋巴细胞活化的抑制作用及机理。方法：分离新鲜人外周血单个核细胞,观察CTLA4-Ig对淋巴细胞转化和混合淋巴细胞培养的作用,用流式细胞术观察CTLA4-Ig对T细胞表面CD25表达的影响,最近通过EMSA方法检测激活T细胞中核蛋白的变化。结果：CTLA4-Ig能够抑制淋巴细胞转化和混合淋巴细胞反应,减低T细胞表面CD25的表达,抑制核内与IL-2表达相关的RE/AP结合因子的活化。结论：CTLA4-Ig通过多种信号途径对T细胞的活化起作用,其机制之一是抑制核内结合于IL-2增强子RE/AP位点的核因子的活化。     

Cytotoxic T lymphocyte antigen 4 immunoglobulin modified dendritic cells attenuate allergic airway inflammation and hyperresponsiveness by regulating the development of T helper type 1 (Th1)/Th2 and Th2/regulatory T cell subsets in a murine model of asthma

T helper type 2 (Th2) and regulatory T cells (T(reg) ) have been postulated to have critical roles in the pathogenesis of allergic asthma. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified dendritic cells (DC-CTLA4Ig) have the potential to reduce Th2 cells and induce T(reg) cells. In the present study, we evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice in an experimental model of asthma. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA for 7 days. Just prior to the first challenge, DC-CTLA4Ig, DCs or DCs infected with DC-green fluorescent protein (GFP) were injected intravenously into mice. The administration of DC-CTLA4Ig reduced airway hyperresponsiveness, relieved asthmatic airway inflammation and decreased the numbers of esosinophils in the BALF in OVA-sensitized/challenged mice. In addition, DC-CTLA4Ig altered the balance of Th1/Th2 cytokine production in the lungs with increased interferon (IFN)-γ levels and decreased interleukin (IL)-4 levels, decreased the percentage of Th2 and increased both the percentage of Th1 and T(reg) cells in the lungs of OVA-sensitized/challenged mice. This research demonstrates that DC-CTL4Ig reduces airway hyperresponsiveness effectively and prevents airway inflammation in OVA-sensitized/challenged mice, which is due most probably to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in the local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/T(reg) cells.     

CTLA4 Silencing with siRNA Promotes Deviation of Th1/Th2 in Chronic Hepatitis B Patients

To determine whether RNA interference （RNAi） could block cytotoxic T-lymphocyte antigen 4 （CTLA4） in human lymphocytes in vitro and promote IFN-T and IL-2 secretions, three small interfering RNAs （siRNAs） were selected based on target specificity sequences of human CTLA4 and transfected into human lymphocytes of chronic HBV patients. As a result, the expression of human CTLA4 mRNA was efficiently suppressed by all the three siRNAs. Compared with negative control （siRNA-co）, siRNA-1 inhibited the expression of CTLA4 most efficiently and was used in the further study. The expressions of IFN-γ and IL-2 were upregulated and the level of IL-4 was almost unchanged in lymphocytes transfected with siRNA-1 compared with the blank control. These results indicated that siRNA-1 led to IFN-γ and IL-2 secretions, which is a main response of Th1/Th2. In a conclusion, RNAi significantly suppressed the expression of human CTLA4 mRNA in human lymphocytes in vitro, and could induce Th1/Th2 response. It could be a new therapeutic strategy for chronic HBV infection.     

CTLA4—lg融合蛋白基因的表达及其生物学功能的初步研究

获得ＣＴＬＡ４全长基因并在真核表达系统中表达。方法通过ＲＴ－ＰＣＲ方法，从ＭＴ２细胞系克隆ＣＴ－ＬＡ４全长基因；经直物重叠和半巢式ＰＣＲ改造，获得含抑瘤素Ｍ信号肽的ＣＴＬＡ４胞外段ＤＮＡ，然后克隆至真核表达载体ｐＩｇ，转染ＣＯＳ和ＣＨＯ－Ｋ细胞表达。结论真核表达体系表达了有生物学活性的ＣＴＬＡ４－Ｉｇ。     

Altered Th1/Th2 balance associated with the immunosuppressive/protective effect of the H-2Ab allele on the response to allo-4-hydroxyphenylpyruvate dioxygenase

The H-2A^b allele exerts a dominant down-regulatory effect on the anti-allo-HPPD (4-hydroxyphenylpyruvate dioxygenase) antibody response, through a hitherto unknown mechanism. In the present study, the allo-variable peptide bound to responder H-2A^k molecules with higher affinity than to H-2A^b ones, arguing against the operation of an affinity hierarchy. Quantitative polymerase chain reaction revealed differences in cytokine mRNA expression between suppressed and high-responder mice. Lymph node cells of responder but not suppressed mice contained high levels of interleukin (IL)-4 mRNA as early as 11 h post-immunization and continued to do so for at least 8 days; this early burst was paralleled by a small burst in transforming growth factor (TGF)-β mRNA level. Differences in IL-12 mRNA were not detected, although an early IL-12 effect could not be excluded. Interferon (IFN)-γ appeared to contribute to the suppression at later time points. Early treatment of responder mice with anti-IL-4 monoclonal antibody (11B11) down-regulated the antibody response. The proliferative T cell response from hyperimmunized mice was reduced but still detectable in the presence of an H-2A^b allele. Thus, in the presence of this allele, the Th1 response is enhanced and that of Th2 cells suppressed, apparently as a result of the bias of H-2A^b-restricted T cells in favor of the Th1 subset.     

Relationship of Th1/Th2 cell balance with the immune response to influenza vaccine during pregnancy

To determine the optimal timing for influenza vaccination in pregnant women, we measured alterations in the types 1 and 2 T helper cell (Th1/Th2) balance during pregnancy, monitored specific immunity to inoculated antigens after vaccination with inactivated influenza vaccine, evaluated the relevance of the Th1/Th2 ratio and immune responses to the vaccination, monitored the maintenance of high antibody titers until delivery and measured the transplacental antibody transfer rate. No significant alterations of the Th1/Th2 balance were noted in the 65% of pregnant women among whom the Th1/Th2 ratio was lower than 9.9% in the first trimester. In those groups with a ratio higher than 10% in the first trimester, there was a tendency for the ratio to decrease as gestation advanced. The efficiency of immunization was not influenced by the Th1/Th2 status or by the stage of gestation. The antibody titer decreased steadily with time from 1 month after vaccination to the time of delivery. Conversely, the transfer rate of antibodies from maternal to fetal blood at the time of delivery increased with the duration of gestation after vaccination. Nevertheless, the antibody titers in both maternal and fetal blood were sufficient to afford protection against infection. Thus, efficient influenza vaccination can be undertaken at any stage of pregnancy. J. Med. Virol. 81:1923–1928, 2009. © 2009 Wiley‐Liss, Inc.