结肠癌组织微环境对树突状细胞的影响

目的 探讨结肠癌组织匀浆上清液模拟肿瘤微环境对人树突状细胞(DC)分化发育的影响,以及血管内皮生长因子A(VEGF-A)在其中所起的作用.方法 制备新鲜结肠癌及癌旁组织匀浆上清液.分离人外周血单个核细胞,含重组人粒细胞.巨噬细胞集落刺激因子(rhGM-CSF)和rhIL-4的1640培养液诱导DC,第2天在此基础上设结肠癌匀浆上清组、癌旁组织匀浆上清组、VEGF-A组及正常DC组,第4天加入结肠癌细胞株SW620抗原,第6天加入脂多糖,第8天收集各组细胞.ELISA检测肿瘤组织匀浆上清液中VEGF-A含量.观察DC形态,流式细胞术检测其免疫表型,RT-PCR检测CD1a表达,CCK-8检测T细胞增殖率及杀伤率.结果 结肠癌组织匀浆上清VEGF-A含量明显高于癌旁组织(P＜0.05);与正常DC组相比,结肠癌匀浆上清组细胞形态明显受到抑制,数目减少,表面抗原表达率明显下降(P＜0.01),混合淋巴细胞反应能力及杀伤力也明显下降(P＜0.01);而VEGF-A组细胞数目及形态与正常DC组相比无明显改变,对所检测的Dc表面抗原并无明显抑制(P＞0.05),但在功能实验中它却起到了明显抑制T细胞增殖及杀伤功能的作用.结论 结肠癌组织匀浆上清液所模拟的微环境对DC的诱导分化及功能有明显的抑制作用,在该过程中VEGF-A起到抑制T细胞免疫功能的作用,但该作用并非通过抑制DC共刺激分子表达而实现.     

肾透明细胞癌组织中树突状细胞与微血管密度之间的关系

目的探讨树突状细胞(DC)的数量及成熟情况与肾透明细胞癌微血管密度(MVD)的相关性。方法收集解放军307医院2010-07/2013-01期间手术的30例肾透明细胞癌患者术后癌组织及癌旁标本,通过免疫组化检测石蜡切片中DC特异性标志CD11c、DC成熟标志MHC-Ⅱ和MVD标志CD34的表达情况并进行分析。结果 30例肾透明细胞癌患者中,癌组织中CD11c阳性率和MVD分别为(93.08±66.14)‰和26.31±11.05个明显高于癌旁组织(25.91±12.29)‰和12.78±5.49个,癌组织中MHC-Ⅱ阳性率为(9.65±3.61)‰明显低于癌旁组织(17.60±6.26)‰。MVD与MHC-Ⅱ阳性率成负相关关系,而与CD11c则无明显相关性。癌组织中各指标的表达与患者性别、年龄、TNM分期之间未见明显相关性。结论肾透明细胞癌癌组织中的血管密度、DC含量高于癌旁组织,而成熟DC含量低于癌旁组织,肿瘤微血管密度与DC成熟度之间存在负相关关系。     

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。     

卵巢癌细胞株冻融抗原负载的树突状细胞诱导CTL抗卵巢癌免疫应答的体外研究

目的研究卵巢癌冻融抗原负载的树突状细胞(dendriticcells,DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤卵巢癌细胞的细胞毒性效应。方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法从卵巢癌细胞系SKOV3中提取的可溶性相关抗原负载DC。流式细胞学检测负载抗原后DC表面各种分化相关抗原的表达,ELISA法检测DC上清中IL12的表达,混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力,MTT法检测抗原负载DC激活的抗原特异性CTL对卵巢癌细胞的杀伤作用。结果与未经抗原负载的DC相比,经卵巢癌抗原负载的DC不仅能更高地表达各种DC分化相关抗原CD1α(73.35%±2.94%vs34.1%±2.35%)、CD83(73.9%±8.46%vs54.68%±3.26%)、CD80(91.95%±2.48%vs52.53%±3.18%)、HLADR(70.05%±2.35%vs48.7%±2.07%)以及CD54(88.9%±5.52%vs71.45%±2.29%),同时具有更强的刺激同种异体T淋巴细胞增殖和IL12分泌的能力(P均<0.05)。此外,卵巢癌细胞SKOV3冻融抗原负载DC激活的CTL在体外对SKOV3的杀伤率为77.35%,显著高于未经抗原负载的DC(P=0.0001)。结论经卵巢癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤卵巢癌细胞的作用。     

CpG-ODN治疗小鼠膀胱肿瘤的实验研究

目的 探讨含CpG序列的寡脱氧核糖核苷酸(CpG Oligodeoxynucleotide, CpG-ODN)对小鼠膀胱肿瘤的治疗作用.方法 建立BTT739荷瘤小鼠动物模型,随机分为CpG-ODN治疗组和PBS对照组,于肿瘤细胞接种后第7、14天给予治疗,每组分两亚组,分别用于测量瘤重、体积及用于观察荷瘤小鼠存活情况.ELISA法检测小鼠血清中IL-12水平.流式细胞仪检测肿瘤组织中浸润性树突状细胞表面共刺激分子CD80、CD86的表达.结果 第2次治疗7d后,平均瘤重CpG-ODN组为(3.30±0.81)g,对照组为(4.50±0.47)g(P<0.01),平均体积CpG-ODN组为(3.57±0.84)cm3,对照组为(4.84±0.58)cm3(P<0.01),CpG-ODN组荷瘤小鼠生存期长于PBS对照组(P<0.05).治疗组及对照组小鼠血清中IL-12浓度分别为(391.5±28.4)pg/mL和(257.2±13.7)pg/mL(P<0.01).肿瘤组织中浸润性树突状细胞(Tumor infiltrating dendritic cell, TIDC)表面CD80、CD86表达水平治疗组分别为(17.75±3.13)%、(18.72±2.79)%,对照组分别为(11.32±1.18)%(P<0.01)、(12.65±1.22)%(P<0.01).结论 CpG-ODN对膀胱肿瘤荷瘤小鼠具有抑瘤效应和生存期延长作用,其机制主要是通过诱导DC成熟、促进Th1型细胞因子分泌、诱导免疫应答向Th1细胞分化.     

转染自体胃癌细胞总RNA的树突状细胞诱导个体化免疫治疗的体外实验

目的探讨转染自体胃癌细胞总RNA的树突状细胞(DC)体外介导抗胃癌的免疫效应。方法制备短期培养的原代胃癌细胞。用rhGM-CSF、rhIL-4和TNF-α体外诱导胃癌患者外周血单个核细胞(PBMC)中DC的发育和成熟,并转染自体肿瘤细胞总RNA,激活自体T细胞产生CTL,用CCK-8试剂盒检测CTL的杀伤活性。应用流式细胞术及混合淋巴细胞培养技术检测DC的免疫功能状态。用ELISA法测定IL-12和INF-γ的水平。结果转染自体肿瘤细胞总RNA的成熟DC,不仅可高表达MHC-I、II类分子及CD80、CD83和CD86协同刺激分子,并可获得高效刺激自体或异体T细胞增殖的能力。转染RNA的成熟DC,分泌IL-12的水平及其刺激产生的CTL培养上清液中INF-γ的水平显著高于单纯成熟DC及未成熟DC;且CTL对自体胃癌细胞的杀伤率显著高于异体组。结论转染自体胃癌细胞总RNA的成熟DC能够体外诱导产生对自体肿瘤细胞具有高度抗原特异性杀伤活性的CTL。     

慢性乙肝患者树突状细胞抗原递呈功能状态的初步研究

目的:研究慢性乙肝患者树突状细胞(DC)抗原递呈功能的改变。方法:从慢乙肝患者和健康人外周血分离单个核细胞,诱导培养出DC,显微镜下观察DC形态并计数,流式细胞仪检测DC表面协同刺激分子(CD80,CD86),MTT法检测DC刺激同种异体淋巴细胞增殖的能力,ELISA法测定培养上清液中IL-12、r-IFN水平。结果:慢乙肝患者外周血单个核细胞诱导培养所获得的DC数量及表面协同刺激分子(CD80,CD86)表达水平、刺激同种异体淋巴细胞增殖能力和细胞因子产生水平均明显低于健康者(P<0.05)。结论:慢乙肝患者DC的数量及抗原递呈、免疫刺激功能状态均处于低下水平。     

激活的人外周血及脐血树突状细胞对肿瘤细胞直接杀伤作用的比较

目的 :探索人外周血或脐带血树突状细胞在干扰素 γ(IFN γ)或细菌脂多糖 (LPS)激活前后对肿瘤细胞杀伤活性的影响。方法 :分离健康供者外周血或脐血单核细胞 ,用重组粒单细胞集落刺激因子 (GM CSF)、白介素 4 (IL 4 )或联合肿瘤坏死因子 (TNF α)将其分别诱导为外周血树突状细胞 (PbDC)及脐血树突状细胞 (CbDC) ,二者分别于诱导第 7天或第 11天在合成培养基中加入LPS或IFN γ继续培养 12小时 ,将其激活。用流式细胞仪检测DC表面共刺激分子的改变 ;同时 ,以Jurkat、HL6 0及Daudi为靶细胞 ,以不同效靶比与DC共同培养 18小时 ,采用51 Cr释放实验检测DC激活前后抗肿瘤活性的差异。结果 :①LPS及IFN γ可上调外周血及脐带血DC表面CD86、CD83的表达 ,以LPS刺激组更明显 ,但对CD1a的表达影响较小。②以PbDC为效应细胞 ,LPS或IFN γ可分别增强DC对Daudi或HL6 0的杀伤活性 ,在效靶比为 2 0 :1时与未加刺激因子对照组(Medium DC)相比差异有显著意义 (P <0 0 1)。然而 ,LPS DC对HL6 0、IFN DC对Daudi则无明显杀伤活性 ;但二者对Jurkat却均有杀伤作用。③以CbDC为效应细胞 ,LPS及IFN γ对其杀伤活性的调节作用与对PbDC的相似。但在未加刺激因子前 ,Cb DC可有效杀伤Jurkat细胞 ,而Medium PbDC对Jurkat细胞未见杀伤     

GM-CSF+IFN-α诱导CML患者骨髓单个核细胞分化的树突细胞的免疫应答

目的:为研究临床注射用IFN-α GM-CSF诱导慢性髓性白血病(CML)患者的骨髓单个核细胞(BMMNC)向树突细胞(DC)的分化及其免疫效应。方法:将取8例CML慢性期患者的BMMNC,接种于用含100 mL/L人AB血清的RMPI1640培养液中,加入不同细胞因子的组合(A组:GM-SCF IL-4;B组:临床注射用IFN-α GM-CSF)后进行培养。培养8 d后,在倒置显微镜下观察DC的形态,用流式细胞术检测细胞上的表面标记。采用异基因混合淋巴细胞反应(allo-MLR)检测DC刺激健康供者外周血淋巴细胞增殖的功能,用MTT比色法检测细胞毒性T淋巴细胞(CTL)特异性杀伤白血病细胞的活性。结果:在不同细胞因子组合诱导下分别培养诱导8 d后的DC,其特征性表面标记(CD80、CD86、HLA-DR、CD83、CD1a)的表达率均高于培养前的DC(P<0.05);但培养8 d的两组DC表面标记的表达率无明显差异。allo-MLR在DC与淋巴细胞之比为1∶10时,B组的刺激指数高于A组(P<0.05)。DC能够刺激异体T淋巴细胞产生特异性的CTL。结论:CML患者的BMMNC在GM-CSF IFN-α诱导下培养后分化的DC可较强地刺激淋巴细胞增殖并可杀伤患者的白血病细胞,有望用于CML的免疫治疗。     

膀胱肿瘤细胞培养上清对树突状细胞的形成及功能的影响

目的: 探讨人膀胱肿瘤细胞BIU -87培养上清对树突状细胞(DC)的生成和功能的影响。方法: 分离培养外周血单个核细胞(PBMC)来源的DC, 实验组加入BIU -87细胞的培养上清液, 以正常培养的DC作为对照组, 应用流式细胞术检测在实验组和对照组不同条件下培养的DC上表面标志的表达, 并用改良的MTT比色法检测和对比两组所培养的DC激活同种异体T细胞的能力及其差别。结果: DC与BIU- 87细胞的上清液共培养后, CD1a、CD83及CD86均呈低表达。实验组与对照组相比较, DC的成熟受到抑制, 对同种异体T细胞激活的作用明显降低 (P<0. 05 )。结论: BIU- 87细胞的上清液可抑制DC的生成及相关表面标志的表达, 这可能是导致膀胱肿瘤患者DC数量减少和功能降低的原因。     

An indexing stage for microscopic scanning of microtitre tray wells

A simple stage has been designed to hold and to move microtitre trays for examination under a low power dissecting microscope. Movement of a ball on a handle from well to well of a reference tray to the left of the stage is mechanically translated into movement from one well to another under the microscope field. Movement can be controlled entirely by touch, and the particular well under the field can be determined from the reference tray position. The flat bottoms of all 96 wells stay in alignment and in focus without further adjustment, enabling rapid scanning of all wells on a tray. The apparatus is particularly useful for the microtitre tray antibody-forming cell plaque assay described by Pike et al. (1982).     

Effect of blood storage on lymphocyte subpopulations

In the evaluation of helper and suppressor T lymphocyte subpopulations testing of the cells may not be carried out until the day following collection of the blood. The possibility that changes occur during overnight storage at 4 degrees C or 22 degrees C has been investigated. No statistical differences in percentages of helper and suppressor T cells between fresh samples and overnight stored samples were found if the blood was kept at 22 degrees C.     

Effect of thymosine on human lymphocyte subpopulations in vitro

During incubation of peripheral blood lymphocytes from healthy blood donors with thymosine there is an increase in the number of high-avidity active lymphocytes with receptors for sheep's red blood cells and of lymphocytes with receptors for the C'3-component of complement and for the FC-fragment of immunoglobulins; the proportion of cap-forming immunoglobulin-positive lymphocytes also increases. The role of T- and B-lymphocyte subpopulations and of their precursors as target cells for the action of thymosine is discussed.Division of Immunology, Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 2, pp. 192–194, February, 1980.     

A yeast surface display system for the discovery of ligands that trigger cell activation

Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vβ domain) were shown to act as ‘pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.     

干扰素调节因子在炎症性疾病中的作用

干扰素调节因子(IRF)家族是能对干扰素的基因表达进行调控的一类转录因子,参与炎症反应、细胞周期和细胞凋亡等生物学过程.IRF4作为一种转录因子,通过影响淋巴细胞的分化及其分泌,干扰素转录调控,病原体免疫反应,细胞因子信号转导,细胞增殖调控,自稳平衡,先天性免疫调控和适应性免疫调控等方面来参与炎症的病理生理过程,成为新...     

Differences in the tissue-specific homing of {alpha}{beta} and {gamma}{delta} T cells to gut and peripheral lymph nodes

Tissue-selective streaming of T cells is considered to be acritical element in the integration of normal immune responsesin intact animals. The results presented in this paper showthat while there were major subsets of gut-homing T cells presentin intestinal lymph, there were considerable differences inthe tissue troplsm of T cell populations circulating in lymphdraining gut and peripheral lymph nodes. Thus, while CD4⁺ cellsreturned preferentially to their tissue of origin, y8 T cellsshowed a strong migratory preference for peripheral lymph nodesregardless of their tissue of origin. In contrast, althougha population of gut-homing CD8⁺ cells was present in Heal lymph,CD8⁺ T cells from peripheral lymph nodes homed equally wellto gut and lymph nodes. There were also considerable differencesin the expression of L-selectln on T cells circulating in thetwo compartments. L-selectin was down-regulated on ßT cells present in Meal lymph but not on T cells which expressedthe highest levels of L-selectin of all T cell subsets. It issuggested that gut-homing ß T cells which have down-regulatedL-selectin are formed in the gut-associated lympnoid tissuesin response to gut antigens while the migratory properties of T cells are ontogenetically determined, independent of antigen.     

Homeostasis of T cell diversity

T cell homeostasis commonly refers to the maintenance of relatively stable T cell numbers in the peripherallymphoid organs.Among the large numbers of T cells in the periphery,T cells exhibit structural diversity,i.e.,theexpression of a diverse repertoire of T cell receptors(TCRs),and functional diversity,i.e.,the presence of T cells atnave,effector,and memory developmental stages.Although the homeostasis of T cell numbers has been extensivelystudied,investigation of the mechanisms underlying the maintenance of structural and functional diversity of Tceils is still at an early stage.The fundamental feature throughout T cell development is the interaction between theTCR and either self or foreign peptides in association with MHC molecules.In this review,we present evidenceshowing that homeostasis of T cell number and diversity is mediated through competition for limiting resources.The number of T cells is maintained through competition for limiting cytokines,whereas the diversity of T cells ismaintained by competition for self-peptide-MHC complexes.In other words,diversity of the self-peptide repertoirelimits the structural(TCR)diversity of a T cell population.We speculate that cognate low affinity self-peptides,acting as weak agonists and antagonists,regulate the homeostasis of T cell diversity whereas non-cognate or nullpeptides which are extremely abundant for any given TCR,may contribute to the homeostasis of T cell number byproviding survival signals.Moreover,self-peptides and cytokines may form specialized niches for the regulation ofT cell homeostasis.Cellular & Molecular Immunology.2005;2(1):1-10.     

Bursa-dependent subpopulations of peripheral B lymphocytes in chicken blood

By selective labeling of juvenile chicken bursal cells with colloidal fluorescein isothiocyanate in situ, the emigration rate of bursal lymphocytes to the periphery was estimated at approximately 0.84% and 0.96% of the peripheral blood lymphocyte (PBL) and splenic B cell pool per hour, respectively. Emigrant bursal cells were found primarily in blood and spleen, with very small numbers migrating to thymus, bone marrow, and gut-associated lymphoid tissues. Emigrant bursal cells expressed high levels of both major histocompatibility complex class II antigen and the Ov alloantigen, a phenotype found on a population comprising approximately 4% of bursal cells from which the bursal emigrants may be derived. Surgical bursectomy at 3 weeks of age revealed that peripheral blood B cells could be divided into three distinct populations. Specifically, 60% of the peripheral blood B cells were short lived with a half-life of about 30 h in the blood. These cells accounted for the great majority of emigrants from the bursa to the peripheral blood. Approximately 35 % of PBL B cells had a half-life of 12 days following bursectomy and comprised cells which did not divide in the periphery. Consequently, we propose that physiological differences between this population and the majority of bursal emigrants are established intrabursally. The remaining PBL B cells, whose relative proportion increases with age from about 5 % of PBL B cells at 2-3 weeks of age, are short lived and are being continually produced from (a) post-bursal site(s) of B cell production.     

Influence of erythrocyte contamination on the optimal phytohaemagglutinin concentration in Chinese hamster lymphocyte cultures

Lymphocyte cultures from the Chinese hamster were made with lymphocytes isolated with Ficoll-Isopaque or with Haemaccel. In some experiments the red blood cells (r.b.c.) that remained after lymphocyte isolation were lysed. Optimum phytohaemagglutinin (PHA) concentration for lymphocyte proliferation depended upon the manner in which the lymphocytes were obtained. Lower PHA concentrations were needed for optimum stimulation of lymphocytes isolated with Ficoll-Isopaque than for lymphocytes isolated with Haemaccel. Lysis of the r.b.c. remaining after isolation of lymphocytes with Haemaccel resulted in a decrease of optimum PHA concentration. The optimum PHA concentration depended strongly on r.b.c. contamination, the higher the r.b.c. contamination the higher the optimum PHA concentration. However, only part of the differences found in optimum PHA concentrations can be attributed to r.b.c. contamination.     

Stability of human lymphocyte differentiation antigens when stored at room temperature

The development of monoclonal antibodies to human differentiation antigens and cytofluorographs have added a new dimension to immune monitoring. However, the technology is such that regionalization will most likely occur. We have demonstrated that human lymphocyte antigens OKT3, OKT4, OKT8 and OKT11 are stable in blood samples stored in ACD at room temperature for less than 72 h. This will allow samples to be analyzed in regional centers.