脐血淋巴细胞凋亡及其影响因素

目的探讨新生儿Ｔ淋巴细胞功能低下的机制。方法以健康成人为对照，采用凋亡细胞计数、片段ＤＮＡ分析、流式细胞分析等方法观察脐血单个核细胞（ＣＢＭＣ）凋亡及其影响因素。结果（１）ＣＢＭＣ自发或经抗ＣＤ３单克隆抗体刺激较成人外周血单个核细胞（ＰＢＭＣ）易发生凋亡；（２）ＣＢＭＣ与成人ＰＢＭＣＢｃｌ２阳性细胞率差异无明显意义；（３）白细胞介素１、干扰素γ对ＣＢＭＣ的凋亡无明显作用，白细胞介素２可部分或全部纠正ＣＢＭＣ凋亡；（４）蛋白抑制剂放线菌酮可迅速诱导ＣＢＭＣ和成人ＰＢＭＣ发生凋亡。结论易于凋亡可能是新生儿Ｔ淋巴细胞发育不成熟的另一重要特征，其凋亡途径为Ｂｃｌ２非依赖途径，白细胞介素２对维持ＣＢＭＣ生存十分重要     

静脉免疫球蛋白对新生儿淋巴细胞CD40与CD40配体表达调节的研究

目的探讨静脉免疫球蛋白（ＩＶＩＧ）调节新生儿Ｂ细胞免疫球蛋白释放的机制。方法采用间接免疫荧光标记法，经流式细胞仪检测ＩＶＩＧ（５ｍｇ／ｍｌ）作用后的脐血淋巴细胞及成人外周血标本的ＣＤ４０与ＣＤ４０配体（ＣＤ４０Ｌ）的表达。结果（１）“休止”状态下脐血单个核细胞（ＣＢＭＣ）可表达类似于成人外周血单个核细胞的ＣＤ４０分子。（２）ＩＶＩＧ不改变ｒＩＬ４激活后的ＣＢＭＣ中ＣＤ４０染色阳性细胞的百分比。（３）佛波醇乙酯及钙离子导入剂活化后的ＣＢＭＣ不表达ＣＤ４０Ｌ。在植物血凝素诱导ＣＢＭＣ后，继续予以ＩＬ２及佛波醇乙酯结合钙离子导入剂的诱导，ＣＢＭＣ始表达ＣＤ４０Ｌ。（４）ＩＶＩＧ可促进活化后ＣＢＭＣ的ＣＤ４０Ｌ表达。结论推测ＩＶＩＧ抑制Ｂ细胞抗体释放的同时，产生了针对Ｔ细胞的反馈信号，刺激了ＣＤ４０Ｌ表达的增加，但在Ｂ细胞上的ＣＤ４０或其他共刺激信号及有关的细胞因子缺乏配合的情况下，ＣＤ４０Ｌ表达的增加并无实际意义     

急性期过敏性紫癜患儿外周血单个核细胞凋亡观察

目的　了解急性期过敏性紫癜（ＨＳＰ）患儿是否存在淋巴细胞凋亡延迟。方法　用荧光染色法及流式细胞技术（ＦＡＣＳ）检测经抗ＣＤ３ 单克隆抗体刺激培养的外周血单个核细胞（ＰＢＭＣ）产生凋亡细胞的百分率。结果　与正常儿童比较，急性期ＨＳＰ患儿ＰＢＭＣ的凋亡细胞百分率显著下降，存在淋巴细胞凋亡延迟现象。结论　推测ＨＳＰ患儿的淋巴细胞凋亡延迟可能为ＨＳＰ的发病机制之一。     

白细胞介素2激活脐血单个核细胞体外抗肿瘤活性的实验研究

为探讨白细胞介素２（ＩＬ－２）激活脐血单个核细胞（ＡＣＢ）对白血病细胞株ＨＬ－６０和Ｋ５６２细胞的杀伤作用以及脐血被激活后能否保持其造血祖细胞生成活性（ＰＣＡ），采用３Ｈ－胸腺嘧啶核苷前标记释放法和半固体培养等方法对其进行了研究。结果：ＩＬ－２激活的脐血细胞具有明显的抗肿瘤活性，且不影响其ＰＣＡ。ＩＬ－２激活脐血细胞的最适条件为：（１）细胞浓度为１×１０６ｍｌ；（２）ＩＬ－２浓度为１０００Ｕ／ｍｌ；（３）效靶比为１００１；（４）体外培养７２小时。脐血经ＩＬ－２激活后免疫表型发生明显变化，产生肿瘤坏死因子（ＴＮＦα）及白细胞介素６（ＩＬ－６），且生成大颗粒淋巴细胞（ＬＧＬ）。ＬＧＬ与Ｋ５６２细胞作用后，后者呈凋亡特征。研究结果为临床应用ＡＣＢ治疗白血病提供了实验依据。     

流式细胞仪分析儿童急性淋巴细胞白血病免疫表型63例

为了了解儿童急性淋巴细胞白血病的免疫表型，应用一组单克隆体及流式细胞仪间接免疫荧光法对６３例初治儿童急性淋巴细胞白血病（ＡＬＬ）免疫分型，Ｔ淋巴细胞、Ｂ淋巴细胞型、Ｔ／Ｂ混合型及裸核ＡＬＬ分别为４１．３％、４６％、９．５％、３．２％，髓系抗原（Ｍｙ）及ＣＤ３４表达率为１７．４％和２８．５％，Ｍｙ＾＋ＡＬＬ中ＣＤ３４表达为５４．５％，显著高于ＭｙＡＬＬ（２３．３％），Ｔ／Ｂ混合型ＡＬＬ对Ｍｙ及ＣＤ３     

下呼吸道感染患儿外周血单个核细胞呼吸道合胞病毒感染的研究

为探讨呼吸道合胞病毒（ＲＳＶ）对外周血单个核细胞（ＰＢＭＣ）的感染情况及感染后细胞免疫的变化，采用免疫组化法对１８例ＲＳＶ性急性下呼吸道感染患儿ＰＢＭＣ内ＲＳＶ及其Ａ、Ｂ亚型抗原进行了检测；采用ＡＰＡＡＰ法、ＭＴＴ比色法和ＥＬＩＳＡ法对Ｔ细胞亚群及Ｔ细胞表面白细胞介素２受体表达、ＰＢＭＣ培养上清液白细胞介素２（ＩＬ－２）活性和可溶性ＩＬ－２受体水平进行了测定。结果显示：１８例ＲＳＶ感染患儿中７例ＰＢＭＣ内可检测到ＲＳＶ抗原；１１例ＲＳＶＡ亚型感染者５例ＰＢＭＣ内均为ＲＳＶＡ亚型阳性，７例ＲＳＶＢ型感染者２例Ｂ亚型阳性；７例恢复期和１０例对照组患儿均为阴性；发病３天以内ＰＢＭＣ中ＲＳＶ抗原阳性者多于３天以后（Ｐ＜０．０５）。ＲＳＶ感染组ＰＢＭＣ内ＲＳＶ抗原阳性者，ＣＤ４细胞比率和ＩＬ－２水平均低于阴性者（ｔ＝２．３８，２．４０，Ｐ值均＜０．０５）。提示：ＲＳＶ性急性下呼吸道感染患儿ＰＢＭＣ可被ＲＳＶ感染，可能由此加重免疫活性细胞损害，导致细胞免疫功能紊乱。     

PMA和IM诱导的新生儿脐带血淋巴细胞增殖反应

为比较新生儿脐带血和成人外周血淋巴细胞对抗ＣＤ３单克隆抗体、ＰＭＡ（Ｐｈｏｒｂｏｌ １２－ｍｙｒｉｓｔａｔｅ １３－ａｃｅｔａｔｅ）和ＩＭ（Ｉｏｎｏｍｙｃｉｎ）刺激的增殖反应,了解新生儿出生时淋巴细胞功能障碍是否与信号传导缺陷有关。方法：流式细胞仪检测脐带血和外周血ＣＤ３＾＋、ＣＤ４＾＋、ＣＤ８＾＋Ｔ淋巴细胞的比率;Ｈ＾３－ＴｄＲ掺入法检测淋巴细胞的增殖反应,结果显示应用抗ＣＤ３单克隆抗体或ＰＭＡ     

新生儿溶血性黄疸患者过氧化状况的初步探讨

测定２０例新生儿Ｇ６ＰＤ缺陷性溶血及１３例新生儿ＡＢＯ溶血病的红细胞ＳＯＤ活性及血浆ＭＤＡ浓度变化，结果显示：（１）ＳＯＤ明显低于对照组、ＭＤＡ明显高于对照组；（２）光疗前、后ＭＤＡ变化无显著性差异，而光疗后ＳＯＤ升高；（３）ＭＤＡ与血清微量胆红素浓度呈正相关。以上结果提示：（１）新生儿溶血性黄疸患者遭受的过氧化损害增强；（２）光疗并不加重对溶血性黄疸新生儿的过氧化损害；（３）ＭＤＡ变化可作为新生     

免疫毒素去除脐血T细胞效率及对造血祖细胞的影响

目的 探讨抗Ｔ细胞免疫毒素体外对脐血Ｔ细胞的清除效率及对造血干／祖细胞的影响，方法（１）用碱性磷酸酶抗碱性酸酶桥联酶标技术法（ＡＰＡＡＰ）分别测定了脐血、骨髓、我周血标本ＣＤ５、ＣＤ８Ｔ细胞的百分率；（２）用单向事淋巴细胞培养（ＭＬＣ）方法比较了外周血与脐血ＭＬＣ增殖反应；（３）分别用ＭＬＣ法和噻唑蓝（ＭＴＴ）比色法；观察了两组抗人白细胞分化抗原的单克隆抗体（ＣＤ５、ＣＤ８）与完整蓖麻毒素（Ｒｉｃ     

维生素A对新生儿单个核细胞IgM生成能力的促进作用

目的探讨维生素Ａ（ＶｉｔＡ）对新生儿脐血单个核细胞ＩｇＭ生成能力的影响。方法应用体外培养试验在培养基中加入一定量的视黄酸（ＲＡ），观察ＶｉｔＡ水平正常组与缺乏组新生儿脐血单个核细胞生成ＩｇＭ的变化。结果ＲＡ能够增加ＳＡＣ刺激下的单个核细胞的ＩｇＭ生成，加ＲＡ后ＶｉｔＡ正常组ＩｇＭ由６６６±４０２μｇ／Ｌ增加到１３８９±９８２μｇ／Ｌ（Ｐ＜０．０１），ＶｉｔＡ缺乏组由３５０±１３６μｇ／Ｌ（增到５８１±２４１μｇ／Ｌ）（Ｐ＜０．０１）。尽管体外加ＲＡ后ＶｉｔＡ缺乏组的ＩｇＭ水平有所上升，但增加量和增加幅度都显著低于ＶｉｔＡ正常组。相关分析的结果表明，新生儿脐血ＶｉｔＡ含量与体外培养加ＲＡ后ＩｇＭ的增加幅度之间存在着一定的正相关关系。结论ＶｉｔＡ对新生儿单个核细胞生成ＩｇＭ的能力具有重要的促进作用     

Killing of human Herpes virus 6-infected cells by lymphocytes cultured with interleukin-2 or -12

BACKGROUND: Human Herpes virus 6 (HHV-6) is a causative agent of exanthema subitum and replicates mainly in lymphocytes. The aim of present study was to investigate cytotoxicity against HHV-6-infected cells by cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). METHODS: Human herpes virus 6-infected and -uninfected lymphocytes were used as target cells. Killing of target cells by CBMC and PBMC was investigated by the chromium release cytotoxicity assay. RESULTS: Freshly isolated CBMC and PBMC did not lyse HHV-6-infected and -uninfected cells. When CBMC and PBMC were cultured with interleukin (IL)-2, HHV-6-infected cells were significantly lysed compared with uninfected cells. Deletion of CD16+ cells by treatment of effector cells with anti-Leu-11b (CD16) antibody with complement reduced cytotoxicity against HHV-6-infected cells and T lymphocyte-rich cells did not lyse HHV-6-infected cells. Treatment of effector cells with anti-Fas ligand antibody and treatment of HHV-6-infected cells with anti-Fas antibody reduced cytotoxicity against HHV-6-infected cells. DNA fragmentation was detected in the supernatant from HHV-6-infected cells cultured with IL-2-activated lymphocytes. Culture of CBMC and PBMC with IL-12 also enhanced cytotoxicity against HHV-6-infected cells. CONCLUSIONS: These data suggest that lymphocytes cultured with IL-2 or IL-12 mediate killing against HHV-6-infected cells and killing of HHV-6-infected cells was through apoptosis. Fas-Fas ligand interaction is one pathway by which HHV-6-infected cells are killed. Killing of HHV-6-infected cells by NK cells activated by cytokines may play a role in the recovery from HHV-6 infection in vitro.     

Effect of indomethacin on IL-1beta, IL-6 and TNFalpha production by mononuclear cells of preterm newborns and adults

The in vitro effect of indomethacin (IM) on the production of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)alpha by cord blood mononuclear cells (CBMC) from preterm newborns was compared to that of peripheral blood MC of adults (PBMC). MC isolated from peripheral blood of adults (PBMC) and cord blood of preterm newborns (CBMC) were incubated without or with lipopolysaccharide (LPS) in the absence or presence of various concentrations of IM. The levels of IL-1beta, IL-6 and TNFalpha in the supernatants were tested by ELISA. MC isolated from preterm newborns were less sensitive to the in vitro effect of IM on IL-1beta and TNFalpha secretion than adult cells. While the spontaneous secretion of IL-1beta and TNFalpha and the production of TNFalpha induced by LPS were significantly increased following incubation of adult PBMC with IM, only the spontaneous synthesis of TNFalpha by CBMC of preterm newborns was affected by this drug. The in vitro production of IL-6 by MC in the two groups was not affected by the addition of IM. It is suggested that IM may affect the preterm's immune response. However, the role of the drug in the frequency and severity of infections in the neonatal intensive care unit needs further investigation.     

Effect of dexamethasone on IL-2 and IL-3 production by mononuclear cells in neonates and adults.

The effect of dexamethasone on interleukin 2 (IL-2) and interleukin 3 (IL-3) production by mononuclear cells in preterm and term infants and adults was evaluated. The capacity of mononuclear cells to produce these cytokines, in preterm infants with bronchopulmonary dysplasia (BPD) and treated with dexamethasone, was compared with that before treatment. Twenty six preterm and 36 term neonates and 24 healthy adults were included in the study. Mononuclear cells isolated from neonatal cord blood (CBMC) and adult peripheral blood (PBMC) were stimulated with phytohaemagglutinin (PHA) in the absence or presence of dexamethasone at concentrations between 10(-8)M and 10(-5)M. IL-2 and IL-3 activities in the supernatant fluids were tested using bioassays. The in vivo effect of the drug on the production of these cytokines by PBMC in 10 preterms was determined before and 24 hours after dexamethasone administration (0.5 mg/kg/day). The production of both cytokines was inhibited in a dose dependent manner. A difference in the sensitivity of mononuclear cells to the inhibitory effect of the drug was found between neonatal cord blood cells and adult PBMC, the former being more sensitive. PBMC from preterm infants treated with dexamethasone for BPD produced significantly less IL-2 and IL-3 as early as 24 hours after the initiation of the treatment (43% and 31%; P < 0.05, respectively). It is concluded that mononuclear cells from preterm and term neonates are more sensitive to the inhibitory effect of dexamethasone on IL-2 and IL-3 production.     

Indomethacin and ibuprofen effect on IL-1ra production by mononuclear cells of preterm newborns and adults

The in vitro effect of indomethacin (IM) and ibuprofen (IB) on the production of the interleukin-1 receptor antagonist (IL-1ra) by cord blood mononuclear cells (CBMC) from preterm newborns was compared to that of peripheral blood mononuclear cells (PBMC) from adults. Mononuclear cells (MC) were incubated with lipopolysaccharide (LPS) in the absence or presence of various concentrations of IM and IB. The level of IL-1ra in the supernatants was tested by ELISA. The results showed a lower ability of MC from preterm newborns to produce IL-1ra as compared with adult cells, supporting the assumption of neonatal immune cell immaturity. IM at pharmacological concentrations caused inhibition of IL-1ra secretion by PBMC from adults whereas IB suppressed the secretion of IL-1ra at higher concentrations only. At the same concentrations neither drug had an in vitro effect on the production of IL-1ra by CBMC of preterm newborns. In conclusion, the lower ability of CBMC of preterm newborns to produce IL-1ra in response to LPS and the absence of an IM and IB effect on the secretion of this cytokine by these cells as compared with PBMC of adults, suggest an underdevelopment of the immune response in preterm newborns.     

Method of birth alters interferon-gamma and interleukin-12 production by cord blood mononuclear cells

Many uncertainties exist regarding the capability of cord blood mononuclear cells (CBMC) to produce cytokines. A number of conflicting reports led us to examine the effects of method of birth on CBMC production of interferon-gamma (IFN-γ), interleukin-4 (IL-4) and interleukin-12 (IL-12). While constitutive production of IL-4 was found in both vaginally and cesarean-delivered infants, constitutive IFN-γ or IL-12 production was found in neither. CBMC from vaginally delivered infants responded to stimulation with concanavalin A/phorbol 12-myristate 13-acetate (Con A/PMA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) with significantly higher levels of IFN-γ than CBMC from unlabored cesarean section (CS) infants. Production of IL-12 was increased in the vaginally delivered group in response to LPS and PHA but not to ConA/PMA. In contrast, mode of delivery was not associated with differences in IL-4 production. These results indicate that mode of delivery significantly alters the capability of CBMC to produce some cytokines and therefore should be taken into account in interpreting fetal/neonatal mononuclear cell function studies.     

激活脐血单个核细胞体外抗白血病细胞株活性研究

目的：探讨植物血凝素(PHA)、白细胞介素-2(IL-2)、抗CD3单克隆抗体(αCD3Ab)激活的脐血单个核细胞(CBMC)体外对K562的杀伤活性,以及外源性刺激因子对CBMC的可溶性白细胞介素-2受体(sIL-2R)含量的影响。方法：采集CBMC分别与不同刺激因子体外短期培养,台盼蓝拒染法测效应细胞的增殖能力,ELISA法检测上清液中sIL-2R的含量, 3 H-TdR释放法测定效应细胞体外对K562靶细胞的杀伤活性。结果：脐血PHA-CD3AK细胞在体外培养3 d后增殖倍数、活化后上清液中sIL-2R的含量显著高于PHA-LAK,CD3AK和LAK细胞(P<0.05)。PHA-CD3AK细胞体外对白血病细胞株K562细胞的细胞毒活性明显高于PHA-LAK,CD3AK和LAK细胞(P<0.05)。结论：脐血单个核细胞经PHA,IL-2和αCD3Ab激活后,可有效形成PHA-CD3AK效应细胞,其增殖活性、细胞毒性均高于PHA-LAK,CD3AK和LAK细胞。     

褪黑素对新生儿脐带血单个核细胞活化增殖的影响

目的探讨褪黑素（melatonin,MLT）对新生儿脐带血单个核细胞（cord blood mononuclear cells,CBMC）活化增殖的影响。方法运用相差显微镜及。H-TdR掺入法观察MLT对CBMC形态学的影响及增殖的调节,并与MLT对成人血单个核细胞（peripheral blood mononuclear cells,PBMC）的影响进行比较。结果CBMC的。H-TdR掺入率随培养基中MLT浓度（50pg／ml～50ng／m1）的增加而增加,在5ng／ml效果最佳。在加入MLT（5ng／m1）的培养基中,经72h培养后,高倍光镜观察下见细胞较培养前明显密集,为较多体积增大的单个核细胞。CBMC培养基中分别加入不同刺激物MLT（5ng／m1）、Interleukin-2（IL-2,50ng／m1）、MLT＋phytohemagglutinin（PHA,5μg/ml）、MLT＋IL-2,其^3H-TdR掺入率（cpm）分别为114327±52863、16087±9006、118360±59207和17682±7391。与细胞悬液组（14133±8688）比较,MLT组和MLT＋PHA组CBMC的。H-TdR掺入率显著增加（t=5．9143,P〈0.001;t=5．5078,P〈0．001）,IL-2组、MLT＋IL-2组CBMC的。H-TdR掺入率无明显变化（t=0．4983,P〉0．05;t=0．9839,P〉0.05）;而MLT组或MLT＋PHA组CBMC的。H-TdR掺入率与培养基中仅加入PHA组（110397±48663）比较,差异无统计学意义（t=0．1730,P〉0．05;t=0．3286,P〉0．05）。各种刺激物加入培养基后,CBMC的^3 H-TdR掺入率均明显高于PBMC。结论MLT不但可诱导PBMC活化增殖,也可诱导CBMC的活化增殖,且其对CBMC的作用强于PBMC。     

Effect of lipid emulsion on IL-2 production by mononuclear cells of newborn infants and adults

The in vitro effect of a lipid emulsion (intralipid) on interleukin-2 (IL-2) production by cord blood mononuclear cells (CBMC) of preterm and term newborn infants was examined and compared to that of peripheral blood mononuclear cells (PBMC) of adults. Intralipid, added at concentrations accepted in clinical practice, caused a dose-dependent inhibition of IL-2 activity tested by bioassay. IL-2 levels, tested by radioimmunoassay (RIA), were found to be reduced only in supernatants derived from CBMC of term infants and not in those derived from MC of preterm infants or adults. The capacity of the IL-2 dependent cell line CTLL-2 to respond to IL-2 was abolished in the presence of intralipid, suggesting an interference with the binding of IL-2 to its receptor on these cells. It is conceivable that administration of intralipid to preterm infants may interfere with the binding of IL-2 to the specific receptors on their activated lymphocytes, with a possible subsequent suppression of their immune response.     

The production of soluble and cellular interleukin-2 receptors by cord blood mononuclear cells following in vitro activation

Cord blood mononuclear cells (CBMC) were investigated for their capacity to generate both cellular and soluble, supernatant interleukin-2 receptors (IL-2R) following cellular activation in vitro. Soluble IL-2R were measured in cell-free supernatants and in detergent-solubilized cell extracts with a "sandwich" enzyme-linked immunosorbent assay. CBMC and adult peripheral blood mononuclear cells were activated with phytohemagglutinin or the murine monoclonal antibody OKT3. CBMC and adult peripheral blood mononuclear cells generated cellular and soluble IL-2R in response to both activators. Peak values for supernatant IL-2R were observed on day 7, while peak values of cell-associated IL-2R occurred on day 5, followed by a decline on day 7. With the exception of supernatant IL-2R production induced by OKT3 stimulation, CBMC produced IL-2R in amounts comparable to adult mononuclear cells. Cord blood plasma also contained amounts of IL-2R comparable to that found in adult sera/plasma. Thus, CBMC appear largely immunocompetent with regard to the expression of IL-2R.     

过敏性紫癜外周血单个核细胞CD40 L表达及其干预研究

目的 观察过敏性紫癜患儿外周血单个细胞表达ＣＤ４０配体及其干预因素,以期探讨在过敏性紫癜发病机制中的作用并指导临床治疗。方法 用流工细胞技术,ＥＬＩＳＡ法及单向免疫扩散法检测观察组３３例患儿ＣＤ４０ Ｌ阳性细胞率,ＩＬ－４,ＩＬ－５及ＩｇＥ,Ａ,Ｇ,Ｍ,并与对照组比较。