卡介苗(BCG)通过诱导IL-12产生和受体表达促进人NK细胞功能

目的:研究卡介苗(BCG)对人自然杀伤细胞(NK)功能的作用及其机制.方法:分离抗结核抗体阴性志愿者外周血PBMC、纯化NK细胞, 分别与BCG、 IL-12、 BCG+IL-12、 BCG+抗IL-12Rβ1 mAb(2B10)培养.利用ELISA方法检测培养上清液IFN-γ、 IL-12p40含量;利用ELISpot方法检测IFN-γ、颗粒酶B产生细胞的频率;利用四甲基偶氮唑盐(MTT)比色法测定杀伤功能.利用流式细胞术检测NK细胞IL-12Rβ1的表达.结果:BCG呈剂量依赖的方式诱导PBMC产生IFN-γ.在BCG刺激条件下, PBMC颗粒酶B分泌细胞数明显高于不加任何刺激剂组(P<0.05).BCG增强PBMC杀伤活性.BCG不能诱导纯化NK细胞产生IFN-γ, 但与IL-12同时刺激则表现出协同作用.纯化NK细胞经BCG刺激后杀伤活性与未刺激相比差异无统计学意义.BCG呈剂量依赖方式诱导PBMC产生IL-12、并促进NK细胞不同亚群表达IL-12Rβ1.2B10抗体抑制BCG对PBMC产生IFN-γ和分泌颗粒酶B的诱导作用.结论:BCG间接地促进NK细胞的生物学活性, 其部分机制是通过诱导单核细胞产生内源性IL-12、并上调NK细胞表达IL-12R.     

TLR4在慢性重型肝炎患者中的表达及意义

Toll样受体4（TLR4）是细胞壁脂多糖（LPS）的特异性受体，可通过特定的细胞信号转导途径介导细胞免疫，以造成机体的损伤。本研究通过检测慢性重型肝炎患者外周血单个核细胞（淋巴细胞、单核细胞）表面的TLR4表达水平，探讨其在慢性重型肝炎中的意义及作用机制。     

IL-10和TNF-α在人外周血单个核细胞体外感染卡介苗中的表达及相关性研究

目的探讨白细胞介素-10（IL-10）、肿瘤坏死因子（TNF-α）在人外周血单个核细胞（PBMCs）体外感染卡介苗（BCG）中的表达及相互作用。方法 PBMCs体外感染BCG后,分别于3、6、8、24h收集细胞应用荧光定量PCR法检测细胞内IL-10和TNF-α的mRNA含量;此外,分别于8、20、32、48h加入anti-IL-10的抗体孵育,收集细胞培养上清,用ELISA法检测上清中IL-10和TNF-α的蛋白含量。结果 PBMCs体外感染BCG后,相对于空白对照组,IL-10和TNF-α在mRNA和蛋白水平的表达量均增高,差异具有统计学意义（P〈0.05）,两者表达量呈现负相关趋势;当加入anti-IL-10的抗体后,TNF-α的蛋白表达量要高于未加抗体组,差异具有统计学意义（P〈0.05）。结论 IL-10和TNF-α在人PBMCs体外感染卡介苗中的表达量增加,IL-10对TNF-α的表达可能具有抑制作用。     

细胞因子及抗-CD3单抗对外周血单个核细胞活化作用

探讨联合应用ＩＦＮ γ、ＩＬ 2及抗 ＣＤ3单抗对分离自健康供血者的外周血单个核细胞 (ＰＢＭＣ)在体外的诱导活化作用 ,并以单独应用ＩＬ 2作比较 ,分析了细胞活化的特征性标志。同时通过动态测定这些标志 ,认识了免疫细胞活化的动态过程。现将研究结果报告如下 :材料与方法试剂 :基因重组人ＩＦＮ γ ,上海克隆生物高技术有限公司产品 ;基因重组人ＩＬ 2 ,中国军事医学科学院基础所及北京中化生物技术研究所联合研制产品 ;ＣＤ3单抗 ,古巴分子免疫中心产品 ;ＭＴＴ及ＤＭＳＯ均为美国Ｓｉｇｍａ公司产品 ;ＦＩＴＣ或ＰＥ标记鼠抗人ＣＤ3…     

诱导活化的外周血单个核细胞THANK表达的初步研究

目的 分析不同刺激下外周血单个核细胞（PBMC）中THANK基因的表达,并克隆全长的人THANK基因,方法 采用将常规方法分离的外周血单个核细胞（PBMC）培养于含100ml/L FCS的RPMI1640中,并以不同浓度的LPS、TNF-α、IL-2、IFN-γ、PHA及PMA刺激不同时间,以RT-PCR法,分析PMBC中THANK基因的转录表达,并克隆相应的全长人THANK基因,结果 RT-PCR分析表明,当用IFN-γ刺激PMBC72h后,可诱导THANK基因明显表达;而IL-2、LPS、TNF-α、PHA和PMA则不能诱导其表达。采用PCR产物克隆的方法,克隆了人THANK基因,并经DNA序列测定证实,结论 克隆得到人THANK基因,并经DNA序列测定证实,结论 克隆得到了人THANK基因,为进一步研究THANK的功能打下了基础。     

CpG-ODN活化人NK细胞的初步研究

目的：研究D型CpG-ODN对人免疫细胞的活化效应。方法：CpG-ODN体外刺激人外周血单个核细胞(PBMC)，ELBA检测培养液IFN-α及IFN-γ的含量；RT-PCR检测PBMC中TLR9的表达水平；MTF法观察活化的NK对K562的杀伤作用。结果：CpG-ODN有效诱导PBMC分泌IFN-α和IFN-γ，增强活化的NK细胞对K562细胞的杀伤作用，且能显著上调TLR9 mRNA的表达。结论：在TLR9的介导下，CpG-ODN能有效激活NK细胞，参与机体免疫调节。     

低剂量γ射线辐照外周血单个核细胞对细胞因子表达的影响

目的:探讨低剂量辐射对外周血单个核细胞细胞因子表达的刺激效应.方法:人外周血单个核细胞采用1 Gyγ射线照射,吸收剂量率为17 Gy/min,并在培养过程中不同时间采用ELISA方法检测上清液中IL-2、TNFα及IFN-γ含量.结果:经γ射线照射24 h后培养上清中IL-2、TNFα及IFN-γ含量明显升高,与对照组比较有明显差异(P<0.05),并随培养时间延长,各细胞因子含量会进一步升高.结论:低剂量辐射对外周血单个核细胞中IL-2、TNFα及IFN-γ表达具有刺激效应.     

原发性肾小球肾炎患者外周血IL—18 mRNA表达的研究

目的 探讨白细胞介素18（IL-18）在原发性肾小球肾炎（PGN）中的作用。方法 采用反转录多聚酶链反应（RT-PCR）及酶联免疫吸附法（ELISA）测定11例正常人及46例不同病程PGN患者外周血单核细胞（PBMC）IL-18 mRNA表达量及血浆中IL-18分泌水平。结果 肾功能代偿期原发性肾小球肾炎患者PBMC IL-18 mRNA表达量及血浆中IL-18的水平与正常对照组无统计学差异,而伴有肾功能损害的PGN患者则显著增高,且与肾功能损害相平行;直线相关分析显示;血浆内IL-18水平与Scr呈正相关、与Ccr呈负相关、与24h尿蛋白排泄量不相关。结论 外周血IL-18不参与PGN早期的发病过程,但当疾病进一步发展至伴有肾功能不全时,IL-18分泌水平相对升高可能在本病的进一步发展中发挥一定的作用。     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.     

Lactate dehydrogenase (LDH) isoenzymes and proliferative activity of lymphoid cells--an immunocytochemical study.

The regulation of cytokine production and T cell proliferation by other cytokines is mandatory in mediating inflammatory responses but the full understanding is far from complete. We have previously reported increased production of IL-2 and IL-2 receptors (IL-2R) in IgA nephropathy. The present study was undertaken to examine other cytokine production during T cell activation in IgA nephropathy. Peripheral blood mononuclear cells (PBMC) from 17 IgA nephritic patients and 14 controls were cultured with phytohaemagglutinin and phorbol myristate acetate for 48 h for maximal cytokine production. IL-2Rs and IL-4 receptors (IL-4Rs) expressed on cultured PBMC were studied by a radioimmunoassay using monoclonal antibodies against these receptors. Although the total cellular IL-2R expression and percentages of T helper and T suppressor cells did not differ between the patients and controls, there was a significant increase in activated T helper cells expressing IL-2R in patients with IgA nephropathy. The total cellular IL-4R expression was elevated in IgA nephritic patients (P less than 0.005). IL-2 production by PBMC was raised in IgA nephritic patients compared with controls (P less than 0.05) but no difference in IL-4 or IL-6 production was observed. The interferon-gamma production by PBMC was significantly increased in patients with IgA nephropathy (P less than 0.025). No correlation was observed between individual cytokine levels. Our data suggest there are selective increases in cytokine production in IgA nephropathy.     

IL-2 receptor gene expression and IL-2 production by human preterm newborns' cells.

IL-2 receptor (IL-2R) gene expression in human umbilical cord blood mononuclear cells (CBMC) of preterm and term newborns was examined following stimulation for 18 h with phytohaemagglutinin (PHA) and compared with that of adult peripheral blood mononuclear cells (PBMC; mothers and control group). mRNA for IL-2R could not be detected in CBMC of preterm infants, whereas the mRNA levels for IL-2R found in full term neonates were similar to those observed in PBMC of adults. IL-2 activity in conditioned medium (CM) of mononuclear cells stimulated with either optimal or suboptimal PHA concentrations for 24 h and 48 h was also determined. At 24 h of stimulation, IL-2 activity found in CM obtained from CBMC of preterm and term newborns was significantly higher than that found in CM of adults' PBMC. A further enhancement of IL-2 activity (six to eight times) was observed in CM of preterm and term cells stimulated for 48 h, whereas no significant difference was found in IL-2 activity in CM from adult cells tested at the two incubation periods. The present findings may provide an additional explanation for the impaired function of the immune system, and the high susceptibility to infections observed in preterm newborns.     

白细胞介素18受体在人类外周血单个核细胞及肾组织表达的研究

目的 :进一步明确白细胞介素 (IL 18)在肾组织免疫性损伤中的作用。方法 :应用半定量逆转录 多聚酶链反应(RT PCR)技术检测 16例正常人 ,16例原发性系膜增生性肾小球肾炎 (MsPGN)患者以及 18例狼疮性肾炎 (LN)患者外周血单个核细胞 (PBMC)白细胞介素 18受体 (IL 18R)α链mRNA的表达量 ,并用免疫组化方法检测 6例正常肾组织 ,16例MsPGN)患者及 18例LN患者肾组织IL 18Rα链蛋白表达量。结果 :MsPGN患者PBMCIL 18RmRNA表达量较正常组有所增高 ,但未达统计学意义 (P >0 0 5 ) ,LN患者PBMCIL 18RmRNA表达量较正常人显著增高 (P <0 0 0 1) ;正常肾组织存在较弱的IL 18R表达 ,MsPGN患者肾组织IL 18R表达量较正常肾组织有所增强 ,但未达统计学差异 ,而LN患者肾组织IL 18R的表达量较正常肾组及MsPGN组均显著增强 (P <0 0 0 1) ,但Pearson相关分析发现LN患者PBMC及肾组织IL 18R表达量均不与血清肌酐 (Scr)水平及 2 4小时尿蛋白排泌量 (2 4h UPE)存在相关关系。结论 :IL 18信号在全身系统及肾组织局部的免疫调节中起一定的作用 ;IL 18在肾功能未严重损害MsPGN患者的发病过程中可能所起作用不大 ;LN患者不但存在IL 18的过量产生 ,而且存在着IL 18过度作用的问题 ,抑制过强的IL 18作用信号可能是LN治疗的新途径。     

Colchicine derivatives inhibit neopterin production in human peripheral blood mononuclear cells (PBMC)

Colchicine is a microtubule disrupting agent, mostly used as treatment in various kinds of inflammatory diseases such as acute familial Mediterranean fever and Behcet's disease, as well as gout. In patients with familial Mediterranean fever treatment with colchicine induces a decline of urinary neopterin concentrations which indicates a decrease of cell-mediated immune activation. In this study, we investigated a potential effect of colchicine on the T cell/macrophage system in vitro. The human myelomonocytic cell line THP-1 and PBMC were treated with colchicine or the colchicine derivative, colcemide, in the presence or absence of 250 U/ml interferon-gamma (IFN-γ) or 10 μg/ml lipopolysaccharide (LPS) for 48 h or 96 h. Colchicine and colcemide increased neopterin/protein production in unstimulated THP-1 cells, but no such effect was apparent in cells stimulated with IFN-γ. By contrast, when PBMC were treated with colchicine or colcemide a significant reduction in neopterin formation was evident in cells without and with prestimulation by IFN-γ or LPS. In parallel, reduced production of IFN-γ was observed in PBMC. These data suggest that colchicine and colcemide are able to inhibit T cell activation within the cellular immune response.     

Expression and Functional Analysis of Toll-Like Receptors of Peripheral Blood Cells in Asthmatic Patients: Implication for Immunopathological Mechanism in Asthma

Background  We investigated the expression profile of toll-like receptors (TLRs) and TLR ligand-activated production profile of asthma-related inflammatory cytokines in asthmatic patients. The expression of TLR1–8 on monocytes, CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, CD19+ B lymphocytes, and dendritic cells, and ex vivo production of cytokines from peripheral blood mononuclear cells activated by TLR ligands were measured by flow cytometry. Discussion  Ex vivo productions of TNF-α, IL-10, and IL-1β by TLR4 and TLR5 ligand LPS and flagellin were significantly lower in asthmatic patients (all P < 0.05). Expression of TLR4 and TLR5 was also found to be significantly lower in asthmatic patients when compared to that of control subjects (all P < 0.05). Therefore, the decreased activation of TLR4 and TLR5 in asthmatic patients might contribute to the immunopathological mechanisms of asthma by reducing the release of Th1 and anti-inflammatory cytokines. Samantha W. M. Lun and C. K. Wong contributed equally to this study.