卡介苗诱导人外周血单个核细胞Th1活化

目的探讨卡介苗(BCG)对人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)Th1活化的调节作用。方法经密度梯度离心法制备PBMCs悬液,用BCG进行体外刺激。WST1法测定BCG对PBMCs增殖能力的影响;ELISA法检测BCG对PBMCs分泌干扰素γ(interferonγ,IFNγ)及IL-4水平的影响;流式细胞术检测BCG对PBMCs Th1活化的影响。结果经BCG体外刺激后,PBMCs的增殖能力、IFNγ的分泌水平及CD4+IFNγ+细胞比例均显著增加(P0.05),IL-4的分泌水平显著下降(P0.05)。结论 BCG可显著诱导PBMCs发生Th1活化,为深入研究BCG的抗肿瘤作用机制奠定了基础。     

3种免疫调节性寡聚脱氧核苷酸佐剂体外免疫活性的比较

目的比较用于乙型肝炎疫苗的3种寡聚脱氧核苷酸(CpG-oligodeoxynueleotide,CpG ODN)佐剂体外的免疫活性。方法分别将CpG ODN 684(沃森公司)、CpG ODN 1018(Dynavax公司)和CpG ODN 7909(Coley公司)序列体外刺激免疫人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)和人树突状细胞系GEN,采用氚-胸腺嘧啶核苷(3H-TdR)渗入法检测人PBMC增殖活力,ELISA法检测GEN细胞中IL-6和IFNα水平。结果 CpG ODN 7909、CpG ODN 1018和CpG ODN 684诱导人PBMC的增殖水平明显高于未刺激的对照组,差异有统计学意义(P0.000 1),其中CpG ODN 684和CpG ODN 7909序列诱导人PBMC的增殖水平相似,略高于CpG ODN 1018序列;3种CpG ODN在体外对免疫细胞均具有明显的活化作用,其中CpG ODN 684和CpG ODN 7909序列体外活化免疫细胞产生的IL-6和IFNα水平相似,但高于CpG ODN 1018序列。结论沃森公司CpG ODN 684序列体外活化免疫细胞功能与Coley公司CpG ODN 7909序列相似,略优于Dynavax公司CpG ODN 1018序列。     

干扰素γ对血小板裂解液扩增的人脐带间充质干细胞免疫调节功能的影响

目的探讨干扰素(interferonγ,IFNγ)对血小板裂解液扩增的人脐带间充质干细胞(umblical cord mesenchymal stem cells,UCMSCs)免疫调节功能的影响。方法采用血小板裂解液(platelet lysate,PL)扩增UCMSCs,通过细胞形态、细胞表型和分化能力检测IFNγ(10 ng/m L)对UCMSCs生物学特性的影响。同时采用ELISA法分析IFNγ对UCMSCs分泌免疫相关因子PGE-2、TGF-β1和IL-6的影响,CCK-8法检测IFNγ对UCMSCs抑制人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)增殖的影响。结果 IFNγ预处理对UCMSCs在细胞形态、细胞表型、细胞成骨和成脂肪的分化能力方面无明显影响(P0.05);IFNγ预处理可显著促进UCMSCs的TGF-β1及IL-6分泌(P0.05),但对PGE-2的分泌无明显影响(P0.05);IFNγ预处理可增强UCMSCs对PBMCs增殖的抑制作用(P0.05)。结论 IFNγ预处理可增强UCMSCs的免疫调节功能。     

荷瘤小鼠骨髓来源的树突状细胞诱导产生的抗肿瘤免疫反应

目的研究荷瘤小鼠骨髓来源的树突状细胞（ＤＣ）是否具有诱导机体产生抗肿瘤免疫反应的能力, 与正常小鼠骨髓来源的ＤＣ有无差异。方法于体外用ｍＧＭ－ＣＳＦ和ｍＩＬ－４分别从正常小鼠和荷Ｃ２６肿瘤的小鼠骨 髓细胞诱导产生ＤＣ,观察它们刺激同种Ｔ细胞、同基因Ｔ细胞增殖的能力,以及由二者免疫所诱导产生的ＣＴＬ的 杀伤活性。结果荷瘤小鼠骨髓来源的ＤＣ与正常小鼠骨髓来源的ＤＣ在刺激同种Ｔ细胞增殖、刺激同基因Ｔ细胞 增殖的能力和诱导产生ＣＴＬ的杀伤活性差异无显著意义。结论荷瘤小鼠骨髓来源的ＤＣ与正常鼠骨髓来源的 ＤＣ具有相似的诱导产生抗肿瘤免疫反应的能力,提示可以从荷瘤机体的ＤＣ前体诱导产生有抗瘤能力的ＤＣ,用于 抗肿瘤免疫治疗。     

人间充质干细胞抑制同种异体人外周血Th17淋巴细胞增殖活性的质量标准体系的建立

目的 构建人间充质干细胞(human mesenchymal stem cells,hMSCs)体外抑制同种异体人外周血Th17淋巴细胞增殖活性的质量标准体系.方法 基于hMSCs与同种异体人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)体外共培养和标准人间充质干细...     

淋巴细胞功能相关抗原-1基因敲除对实验性自身免疫性脑脊髓炎小鼠引流淋巴结T细胞活性的影响

目的探讨淋巴细胞功能相关抗原-1(lymphocyte function-associated antigen-1,LFA-1)基因敲除对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型小鼠引流淋巴结T细胞活性的影响。方法采用髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG)35-55多肽诱导LFA-1a链CD11a基因敲除的C57BL/6小鼠及野生型C57BL/6小鼠,建立EAE小鼠模型,取MOG诱导后21 d EAE模型小鼠脊髓颈段切片,HE染色和LuxolFastBlue染色,观察小鼠脊髓组织学变化;于MOG诱导后7、14、21d处死小鼠,收集腹股沟引流淋巴结CD4+T淋巴细胞,加入终浓度为1μg/ml的MOG35-55多肽刺激48 h后,应用BrdU试剂盒检测细胞增殖情况;于MOG诱导后4、7、10、14、21 d处死小鼠,收集腹股沟引流淋巴结细胞,加入终浓度为1μg/ml的MOG35-55多肽刺激48 h后,进行细胞内细胞因子染色,观察分泌IFNγ和IL-17的T细胞比例。结果 MOG诱导的野生型小鼠脊髓出现明显淋巴细胞浸润及广泛的脱髓鞘变化,而LFA-1基因敲除小鼠无明显变化;在EAE疾病早期(7 d),LFA-1基因敲除可减少淋巴细胞向引流淋巴结聚集,降低MOG体外刺激T细胞增殖能力,分泌IFNγ及IL-17的T细胞比例也较野生型C57BL/6小鼠明显降低(P0.05);而在EAE疾病缓解期(21 d),LFA-1基因敲除小鼠淋巴细胞聚集及T细胞增殖能力与野生型C57BL/6小鼠相比无明显差异(P0.05),分泌IFNγ及IL-17的T细胞比例高于野生型C57BL/6小鼠(P0.05)。结论在EAE疾病发生早期,LFA-1可能起到了关键性的作用,LFA-1有可能成为自身免疫性疾病治疗的重要分子靶点。     

选择性去除表达CD25 、CD69 的同种异体反应T细胞的实验研究

目的 探讨体外选择性去除表达CD25 、CD69 的同种异体反应T细胞协调移植物抗宿主病和移植物抗肿瘤的可能性.方法 流式细胞仪检测初次单向混合淋巴细胞反应CD25 、CD69 表达情况,磁性细胞分离系统在细胞表达高峰去除CD25 、CD69 同种异体反应rr细胞.MTT法在各时间点检测不同抗原组再次混合淋巴细胞反应的细胞增殖情况.结果 再次单向混合淋巴细胞反应显示明显降低了对相同刺激原的反应性(48.82±13.71)%,保留大部分对无关刺激原的反应性(65.47±9.84)%和肿瘤刺激原的反应性(84.83±3.65)%.结论 体外选择性去除表达CD25 、CD69 的同种异体反应T细胞可以协调移植物抗宿主病和移植物抗肿瘤.     

卡介苗与结核分枝杆菌早期分泌靶抗原刺激人γδT细胞分泌细胞因子能力的比较

目的比较卡介苗(bacillus Calmette-Guerin vaccine,BCG)与结核分枝杆菌早期分泌靶抗原(early secreted antigenic target-6,ESAT-6)刺激人外周血γδT细胞分泌细胞因子的能力。方法采用Ficoll密度梯度离心法分离健康人外周血单核细胞(peripheral blood mononuclear cell,PBMC),加入Anti-human gamma-delta TCR-FITC抗体标记γδT细胞,上流式细胞仪进行分离纯化;分别用BCG和ESAT-6刺激γδT细胞,同时,以不加任何刺激因子的γδT细胞作为空白对照,分别于刺激培养后第1、3、6、9和12天取上清,采用ELISA试剂盒检测IL-17、TNF-α及IFNγ的分泌水平;上流式细胞仪检测γδT细胞的增殖水平。结果γδT细胞占PBMC的4.83%,其纯度为88.90%;与空白对照组比较,BCG和ESAT-6刺激的γδT细胞分泌的IL-17、TNF-α及IFNγ水平均明显升高,且ESAT-6组明显高于BCG组(P均<0.05);与空白对照组相比,BCG组和ESAT-6组γδT细胞数量明显增加,且ESAT-6组较BCG组增加明显(P均<0.05)。结论 BCG和ESAT-6均可刺激γδT细胞增殖,并大量分泌IL-17、TNF-α及IFNγ,且ESTA-6的刺激作用强于BCG。     

黄芪多糖对胃癌细胞上清液中培养的树突状细胞分化成熟及功能的影响

目的探讨黄芪多糖对胃癌细胞SCG-7901上清液中培养的树突状细胞(Dendritic cells,DC)分化成熟及功能的影响,分析黄芪多糖抗癌的作用机制。方法将人外周血分离的单核细胞加入含重组人粒细胞-巨噬细胞集落刺激因子(rhGMCSF)和重组人白细胞介素的培养液中,随机分为空白组(以RPMI1640培养液培养)、干预组(以黄芪多糖干预后的胃癌细胞上清液培养)、对照组(以胃癌细胞上清液培养)。混合培养48h后,显微镜下观察DC成熟过程中的形态学变化,流式细胞术检测DC的表型(CD40、CD80),混合同种淋巴细胞增殖反应(Mixed lymphocyte reaction,MLR)检测DC的增殖效应。结果与对照组比较,空白组和干预组DC的CD40和CD80的表达均明显增加(P<0.05),刺激同种淋巴细胞增殖效应明显增强(P<0.05);干预组DC CD40和CD80的表达阳性率及刺激同种淋巴细胞增殖效应均明显低于空白组(P<0.05)。结论在体外,黄芪多糖可有效对抗胃癌细胞上清液引起的对DC分化成熟和功能的抑制。     

绿脓杆菌及其鞭毛蛋白联合rhIL-12体外对慢性乙型肝炎患者细胞免疫功能的影响

目的探讨绿脓杆菌及其鞭毛蛋白联合rhIL-12体外对慢性乙型肝炎患者细胞免疫功能的影响,为rhIL-12作为佐剂应用于临床提供理论依据。方法取健康人及慢性乙型肝炎患者外周血单个核细胞(PBMCs),分别与绿脓杆菌、鞭毛蛋白、rhIL-12、绿脓杆菌+rhIL-12、鞭毛蛋白+rhIL-12孵育,ELISA法检测细胞培养上清中IFNγ的含量。结果绿脓杆菌和鞭毛蛋白组只诱导产生低水平的IFNγ,rhIL-12组诱导产生IFNγ的水平较阴性对照组显著提高;而绿脓杆菌+rhIL-12组、鞭毛蛋白+rhIL-12组诱导产生IFNγ的水平显著高于绿脓杆菌、鞭毛蛋白和rhIL-12组,差异均有统计学意义,且rhIL-12的作用呈剂量依赖性。结论绿脓杆菌及其鞭毛蛋白联合rhIL-12体外能显著提高健康人和慢性乙型肝炎患者PBMCs产生IFNγ的水平,增强其细胞免疫应答。     

干扰素治疗慢性粒细胞白血病微量残留病疗效分析

应用染色体技术和聚合酶链反应方法检测干扰素治疗慢性粒细胞白血病完全缓解后微量残留病28例的疗效。发现治疗后2例Ph'染色体阴转，长期应用干扰素组Ph'染色体阳性率下降，1例bcr／ab1mRNA转为阴性。而短期应用干扰素组无Ph'染色体变化，或bcr／ab1mRNA阴转。按目前我们应用干扰素的剂量、疗程，很难达到有效清除bcr／ab1mRNA阳性细胞的目的。     

6Ckine修饰的树突状细胞促进T淋巴细胞增殖和分化的作用

目的研究次级淋巴组织趋化因子(6Ckine)修饰的树突状细胞(DC)对T淋巴细胞增殖和分化的影响。方法用携带人6Ckine基因的重组复制缺陷型腺病毒(Ad-6Ckine)感染人外周血单个核细胞来源的DC,检测Ad-6Ckine-DC对6Ckine的表达及细胞因子分泌的影响,并观察其吞噬功能和表型的变化及对自身T淋巴细胞的趋化作用。用结肠癌LoVo细胞抗原致敏Ad-6Ckine-DC,将该DC与自身T淋巴细胞共同培养,分别用3H掺入法、RT-PCR和ELISA检测Ad-6Ckine-DC对T淋巴细胞增殖和分化的影响。结果在Ad-6Ckine转染后24h内,DC的吞噬功能几乎不受影响。转染的6Ckine基因能在DC中表达,表达的6Ckine能促进其表达CD83和CCR7,上调RANTES的表达。Ad-6Ckine-DC对自身T淋巴细胞有明显的趋化作用,抗原致敏的Ad-6Ckine-DC能显著促进T淋巴细胞的增殖,并增强其表达T-bet和IL-2的能力。结论6Ckine基因的修饰能在一定程度上促进DC的成熟,并募集T淋巴细胞于DC周围,有利于DC向T淋巴细胞传递抗原和第二信息,增强DC促进T淋巴细胞增殖的作用并使其向Th1分化,诱导细胞免疫,将成为制备肿瘤疫苗的一种良好选择。     

稳定表达BCR/ABL-T315I的小鼠BP210-T315I细胞株的建立

目的构建能稳定表达T315I的BCR/ABL的小鼠BP210-T315I细胞株,并观察该细胞株对酪氨酸激酶抑制剂STI571的敏感性。方法采用PCR法将重组逆转录病毒载体MIGR-P210中的abl基因第944位碱基C定点突变为T,再将该位点突变成功的逆转录病毒载体转染到包装细胞内,收集病毒,感染BaF3细胞,经筛选和亚克隆获得稳定表达T315I的BCR/ABL蛋白的小鼠BP210-T315I细胞。采用RT-PCR、DNA测序和Western blot鉴定abl基因第944位碱基是否发生突变;MTT法检测BP210-T315I细胞对STI571的耐药性。结果 T315I的bcr/abl基因已整合到BaF3细胞基因组中,B210-T315I细胞能稳定表达T315I的bcr/abl基因与蛋白;与BaF3-P210细胞株相比,经STI571处理后,BP210-T315I细胞对STI571明显耐药,其耐药倍数约为100。结论已成功构建稳定表达小鼠T315I的转化细胞株,该细胞株具有对STI571耐药的恶性表型特征。     

Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells

Irradiated murine induced-pluripotent stem cells (iPSCs) elicit the antitumor response in vivo. However, it is unclear whether human iPSCs would elicit antitumor effects. In the present study, we investigated the capability of human iPSC lysate (iPSL)-pulsed dendritic cells (DCs) (iPSL/DCs) to induce cancer-responsive cytotoxic T lymphocytes (CTLs) in vitro. iPSCs and DCs were induced from peripheral blood mononuclear cells isolated from a human leukocyte antigen (HLA)-A33 homozygous donor. The iPSL was pulsed with immature DCs, which were then stimulated to allow full maturation. The activated DCs were co-cultured with autologous CTLs and their responses to SW48 colorectal carcinoma cells (HLA-A32/A33), T47D breast cancer cells (HLA-A33/A33), and T98G glioblastoma cells (HLA-A02/A02) were tested with enzyme-linked immunospot (ELISPOT) assays. Comprehensive gene expression analysis revealed that the established iPSCs shared numerous tumor-associated antigens with the SW48 and T47D cells. Immunofluorescent analysis demonstrated that the fluorescent-labeled iPSL was captured by the immature DCs within 2 h. iPSL/DCs induced sufficient CTL numbers in 3 weeks for ELISPOT assays, which revealed that the induced CTLs responded to SW48 and T47D cells. Human iPSL/DCs induced cancer-responsive CTLs on HLA-A33-matched cancer cells in vitro and could be a promising universal cancer vaccine for treating and preventing cancer.     

特异性抗原致敏的DC-CIK细胞对肿瘤细胞的杀伤效应

目的检测特异性抗原致敏的DC-CIK细胞对恶性肿瘤细胞的杀伤效应。方法采用肿瘤抗原致敏的DC与CIK细胞共培养,分析其免疫表型,并用MTT染色法检测其对肿瘤细胞的杀伤力。结果所获得DC-CIK细胞的CD3异质性T细胞群,对肾透明细胞癌786-0和前列腺癌PC-3细胞的杀伤率分别为70.64%和65.65%。结论DC-CIK细胞对肾透明细胞癌786-0细胞和前列腺癌PC-3细胞具有较强的杀伤效应。     

Caffeic Acid Enhances the Anti-Leukemic Effect of Imatinib on Chronic Myeloid Leukemia Cells and Triggers Apoptosis in Cells Sensitive and Resistant to Imatinib

Among the phenolic acids tested on the K562 cell line, a model of chronic myeloid leukemia (CML), caffeic acid (CA) was biologically active on sensitive and imatinib (IM)-resistant cells at micro-molar concentration, either in terms of reduction of cell proliferation or triggering of apoptosis. The CA treatment provoked mitochondrial membrane depolarization, genomic DNA fragmentation and phosphatidylserine exposure, hallmarks of apoptosis. Cell cycle analysis following the treatment with comparable cytotoxic concentrations of IM or CA showed marked differences in the distribution profiles. The reduction of cell proliferation by CA administration was associated with increased expression of two cell cycle repressor genes, CDKN1A and CHES1, while IM at a cytotoxic concentration increased the CHES1 but not the CDKN1A expression. In addition, CA treatment affected the proliferation and triggered the apoptosis in IM-resistant cells. Taken together, these data suggested that CA induced the anti-proliferative effect and triggered apoptosis of CML cells by a different mechanism than IM. Finally, the combined administration of IM and CA at suboptimal concentrations evidenced a synergy of action in determining the anti-proliferative effect and triggering apoptosis. The ability of CA to potentiate the anti-leukemic effect of IM highlighted the nutraceutical potential of CA in CML.     

Peripheral Blood T Cells of Patients with IPAH Have a Reduced Cytokine-Producing Capacity

Pulmonary arterial hypertension (PAH) is rare disease that is categorized as idiopathic (IPAH) when no underlying cause can be identified. Lungs of most patients with IPAH contain increased numbers of T cells and dendritic cells (DCs), suggesting involvement of the immune system in its pathophysiology. However, our knowledge on circulating immune cells in IPAH is rather limited. We used flow cytometry to characterize peripheral blood DCs and T cells in treatment-naive IPAH patients, compared with connective-tissue disease-PAH (CTD-PAH) patients and healthy controls (HCs). At diagnosis, T-helper (Th) cells of IPAH patients were less capable of producing TNFα, IFNγ, IL-4 and IL-17 compared to HCs. IPAH patients showed a decreased frequency of Th2 cells and significantly enhanced expression of the CTLA4 checkpoint molecule in naive CD4⁺ T cells and both naive and memory CD8⁺ T cells. Frequencies and surface marker expression of circulating DCs and monocytes were essentially comparable between IPAH patients and HCs. Principal component analysis (PCA) separated IPAH patients—but not CTD-PAH patients—from HCs, based on T-cell cytokine profiles. At 1-year follow-up, the frequencies of IL-17⁺ production by memory CD4⁺ T cells were increased in IPAH patients and accompanied by increased proportions of Th17 and Tc17 cells, as well as decreased CTLA4 expression. Treatment-naive IPAH patients displayed a unique T-cell phenotype that was different from CTD-PAH patients and was characterized by reduced cytokine-producing capacity. These findings point to involvement of adaptive immune responses in IPAH, which may have an implication for the development of therapeutic interventions.