CD4单克隆抗体改变新生小鼠胸腺细胞亚群的分布和功能

新生期小鼠（３～７天）腹腔注入ＣＤ４ＭｃＡｂ，可引起胸腺细胞表型和功能变化。胸腺细胞表型分析显示：胸腺细胞总数、ＤＰ和ＣＤ４ＳＰ细胞明显减少；ＣＤ８ＳＰ细胞数增加；ＤＮ细胞数目变化不大。上述变化与ＣＤ４ＭｅＡｂ和胸腺细胞表面的ＣＤ４分子的结合有关。功能试验表明：实验组小鼠胸腺细胞对有丝分裂素ＣｏｎＡ、ＰＷＭ的反应性降低，混合淋巴细胞反应（ＭＬＲ）也明显低于对照组。但是胸腺细胞对异型细胞的杀伤作用则高于对照组。功能试验的结果与表型的变化是吻合的，有剂量依赖性。ＣＤ４ＭｃＡｂ引起的胸腺细胞表型和功能的变化是可以恢复的，一般于注射ＣＤ４ＭｃＡｂ后１０天，胸腺细胞亚群分布恢复或接近正常，而功能则恢复较慢。     

CD4单克隆抗体改变新生小鼠胸腺亚群的分布和功能

新生期小鼠腹腔注入ＣＤ４，ＭｃＡｂ、可引起胸腺细胞表型和功能变化。胸腺细胞表型分析显示；胸腺细胞总数，ＤＰ和ＣＤ４ＳＰ细胞明显减少；ＣＤ８ＳＰ细胞数增加ＤＮ细胞数目变化不大。上述变化与ＣＤ４ＭｃＡｂ和胸腺细胞表面的ＣＤ４分子的结合有关。功能试验表明：实验组小鼠胸腺细胞对有丝分裂素ＣｏｎＡ，ＰＷＭ的反应性降低，混合淋巴细胞反应也明显低于对照组。     

川崎病患者外周血淋巴细胞凋亡减少的初探

目的：探讨川崎病（ＫＤ）的免疫发病机制。方法：对２６例ＫＤ患者和２０名正常儿童外周血单个核细胞（ＰＢＭＣ）经ａｎｔｉ－ＣＤ３诱导体外培养不同时间的凋亡进行计数凋亡细胞百分率和片段ＤＮＡ分析。结果：ＫＤ患者凋亡细胞百分率和片段ＤＮＡ出现时间较正常对照降低（Ｐ〈０．００１）和延迟，ＰＢＭＣ体外培养产生ＩＬ－６水平较正常对照显著升高（Ｐ〈０．００１）；加抗ＩＬ－６单抗培养或静脉注射免疫球蛋白（ＩＶＩＧ）     

AC－cAMP－PKA途径对淋巴细胞DNA合成的调节作用

以ＣＤ３ＭｃＡｂ为激动剂，以淋巴细胞体外ＤＮＡ合成为研究手段，探讨了ＡＣ－ｃＡＭＰ－ＰＫＡ信号途径在ＣＤ３ＭｃＡｂ诱导的淋巴细胞活化中的意义。研究结果表明ＡＣ、ｃＡＭＰ和ＰＫＡ在决定细胞对外界刺激的反应中起着重要作用。在淋巴细胞活化早期细胞内ｃＡＭＰ出现一过性升高，随着细胞活化增殖，ｃＡＭＰ降至正常水平以下。活化ＡＣ、升高细胞内ｃＡＭＰ水平可显著降低ＣＤ３ＭｃＡｂ诱导的淋巴细胞ＤＮＡ合成，而ＰＫＩ却能在一定程度上促进淋巴细胞活化增殖。     

抗细菌脂多糖核心糖脂域单克隆抗体在实验性菌血症中的保护作用

以小鼠菌血症模型对抗核心糖脂域ＭｃＡｂ（ＥＬ１、ＥＬ３和３Ｈ４）的免疫保护的效果进行了研究，结果表明，（１）３株ＭｃＡｂ能显著提高受Ｅ．ｃｏｌｉ攻击小鼠的存活率（Ｐ〈０．０５）；（２）ＥＬ３和３Ｈ４还分别有降低３ＬＤ５０绿脓杆菌和５ＬＤ５０鼠伤寒沙门氏菌感染小鼠病死率的作用（Ｐ〈０．０５）；（３）ＥＬ３和３Ｈ４对受５ＬＤ５０Ｅ．ｃｏｌｉ和１０ＬＤ５０绿脓杆菌攻小鼠有一定的协同保护作用；（４）在ＥＬ     

IL-6对急性期川崎病患者外周血淋巴细胞凋亡的影响

目的拟通过对急性期川崎病（ＫＤ）患者外周血淋巴细胞凋亡的观察，进一步探讨ＫＤ的免疫发病机理。方法２６例ＫＤ患者和２０名正常儿童外周血单个核细胞（ＰＢＭＣ）经抗－ＣＤ３诱导处理，部分用ＩＬ－６单抗处理，之后培养不同时间（０，１２，２４，４８，７２小时）测定凋亡细胞百分率。结果ＫＤ患者凋亡细胞百分率和ＤＮＡ片段化均较正常对照降低（Ｐ＜０．００１）和延迟。ＰＨＡ刺激ＰＢＭＣ培养上清ＩＬ－６水平较正常对照明显升高（Ｐ＜０．００１）。加抗ＩＬ－６的单抗培养或静脉注射免疫球蛋白可显著降低ＩＬ－６水平，凋亡细胞百分率降低和ＤＮＡ片段化延迟发生逆转。结论ＫＤ患者ＰＢＭＣ存在有凋亡降低或延迟，其机理可能与ＫＤ患者异常增高的ＩＬ－６有关     

广枣总黄酮对小鼠胸腺细胞凋亡及腺苷脱氨酶(ADA)活性的影响

目的研究蒙药广枣总黄酮（ＴＦＣ）对地塞米松（ＤＥＸ）诱导的小鼠胸腺细胞凋亡及胸腺细胞内腺苷脱氨酶（ＡＤＡ）活性降低的免疫调节作用。方法将小鼠分为ＤＥＸ组、ＴＦＣ组、ＴＦＣ＋ＤＥＸ组和正常对照组，采用光镜（计凋亡率，数据经方差分析）、电镜（形态学观察）。ＤＮＡ琼脂糖凝胶电泳及紫外分光光度比色法（吸光度值经ｔ检验处理）在培养３，６，９，１２，２４小时动态观察下，研究ＴＦＣ对胸腺细胞凋亡及ＡＤＡ活性的影响。结果在不同的培养时间均看到ＴＦＣ抑制ＤＥＸ诱导的胸腺细胞凋亡（Ｐ＜０．０１）及ＴＦＣ的促胸腺细胞分裂、增殖现象，但ＴＦＣ对细胞培养产生的自发凋亡似无抑制作用（Ｐ＞０．０５）；ＴＦＣ可促进胸腺萎缩小鼠的胸腺细胞内ＡＤＡ活性恢复（Ｐ＜０．０１）。结论ＴＦＣ有提高机体免疫功能作用，这对临床上应用糖皮质激素导致的免疫缺陷、重症感染及肿瘤等的治疗提供了实验依据。     

AC—cAMP—PKA途径对淋巴细胞DNA合成的调节作用

以ＣＤ３ＭｃＡｂ为激动机，以淋巴细胞体外ＤＮＡ合成的研究手段，探讨了ＡＣｃＡＭＰ－ＰＫＡ信号途径在ＣＤ３ＭｃＡｂ诱导的淋巴细胞活化中的意义，研究结果表明ＡＣ，ｃＡＭＰ和ＰＫＡ在决定细胞对外界刺激反应中起着重要作用，在淋巴细胞活化早期细胞内ｃＡＭＰ出现一过性升高，随着细胞活化增殖，ｃＡＭＰ降至正常水平以下，活化ＡＣ，升高细胞内，ｃＡＭＰ水平可显著过性升高，随着细胞活化增殖，ｃＡＭＰ降至正常水平以下，     

淋巴细胞经TCR－CD3活化增殖作用的分析

本文探讨了抗ＣＤ３单抗诱导的淋巴细胞活化增殖及有关影响因素。实验结果表明：①淋巴细胞内钙升高是淋巴细胞活化增殖的重要条件，ＣＤ３ＭｃＡｂ引起的早期胞浆游离钙迅速升高主要由内质网释放钙离子所致，而淋巴细胞增殖不仅需要细胞内钙释放，还需要细胞外钙内流；②ＧＴＰ结合蛋白是淋巴细胞激活过程的一重要环节，经Ｇ蛋白作用物霍乱毒素作用后，淋巴细胞ＤＮＡ合成显著降低；③新霉素和ＰＳＳ可抑制ＰＬＣ和ＰｋＣ的活性，对淋巴细胞ＮＤＡ合成造成剂量依赖性抑制作用。此外，抗ＣＤ３ＭｃＡｂ诱导的淋巴细胞ＤＮＡ合成需要辅佐细胞的存在，高度纯化的Ｔ细胞对ＣＤ３ＭｃＡｂ的刺激不发生增殖反应。     

MTEC 1分泌的趋化因子引起特定亚群胸腺细胞的定向迁移

分析胸腺髓质上皮样细胞系ＭＴＥＣ１分泌的化学趋化因子对胸腺细胞亚群的趋化作用。方法以抗体加补体杀伤结合免疫磁珠及ｐａｎｎｉｎｇ法，将小鼠胸腺细胞分离纯化，获得ＣＤ４＋ＣＤ８＋（ＤＰ），ＣＤ４－ＣＤ８－（ＤＮ），ＣＤ４＋ＣＤ８－（ＣＤ４ＳＰ）及ＣＤ４－ＣＤ８＋（ＣＤ８ＳＰ）四亚群细胞，用Ｂｏｙｄｅｎ小室分析ＭＴＥＣ１┐ＳＮ对四群胸腺细胞的趋化作用。结果ＭＴＥＣ１┐ＳＮ对ＤＰ及ＣＤ４ＳＰ胸腺细胞有趋化活性（ＣＩ＝６．６±１．０及６．１±１．８）；对ＣＤ８ＳＰ细胞有中度趋化活性（ＣＩ＝３．２±１．０）；对ＤＮ趋化活性微弱（ＣＩ＝１．３±０．６）。化学趋化因子ＭＣＰ┐１纯品对ＣＤ４ＳＰ胸腺细胞显示强趋化活性（ＣＩ＝５．６），对ＤＮ胸腺细胞则无可测出趋化活性。结论ＭＴＥＣ１分泌的化学趋化因子对ＤＰ，ＣＤ４ＳＰ及ＣＤ８ＳＰ胸腺细胞有显著趋化作用，对ＤＮ胸腺细胞几乎无趋化作用。提示此类化学趋化因子有趋使胸腺发育中后期阶段的细胞向胸腺髓质区迁移和定位的作用。     

CD4+CD8+ thymocytes are susceptible to DNA fragmentation induced by phorbol ester, calcium ionophore and anti-CD3 antibody

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18-24 h resulted in 70%-80% fragmentation of DNA into 180-200-bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4+CD8+ cells and in a portion of CD4-CD8+ cells. To investigate whether DNA cleavage is also inducible via antigen-specific receptors, thymocytes were incubated in wells precoated with anti-CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti-CD3 antibody. In this anti-CD3-induced DNA degradation, CD4+CD8+ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti-CD3-induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti-CD3-coated wells revealed that CD4+CD8+ cells were preferentially decreased. Further experiments with purified CD4+CD8+ cells, however, could not define a clear-cut increase of DNA fragmentation when isolated CD4+CD8+ cells were stimulated with anti-CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon-gamma to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.     

高浓度抗CD3单抗诱导人外周血单个核细胞凋亡及细胞因子对其影响

使用抗CD3单抗诱导新鲜分离的人外周血单个核细胞(PBMC)凋亡,同时观察细胞因子对其影响。18h后电镜发现处理组发生典型凋亡改变,DNA电泳出现典型阶梯状条带。TUNEL法流式细胞仪检测发现处理组凋亡发生率39.6％,显著高于对照组(<0.01)。IFN-7/TNF-a可显著增强抗CD3单抗诱导的凋亡。IL-1～IL-3、TNF-=对凋亡无明显影响。CsA可抑制抗CD3单抗诱导的凋亡,但加入IFN-y则可完全逆转这种作用。结果表明抗CD3单抗可诱导PBMC发生凋亡,IFN-Y、TNF-与抗CD3有协同作用。     

人参抑制小鼠胸腺细胞凋亡的实验研究

用大肠杆菌脂多糖（ＬＰＳ）所致小鼠胸腺细胞凋亡模型和琼脂糖凝胶电泳、流式细胞术及免疫组织化学等方法，研究不同浓度高丽人参水提取物体内对小鼠胸腺细胞的影响。结果表明，每只小鼠腹腔注射１０￣２０ｍｇ人参提取物可防止ＬＰＳ所致小鼠体重及胸腺减重，提高胸腺细胞存活力，抑制胸腺细胞ＤＮＡ降解和促凋亡蛋白Ｂａｘ的过量表达，免疫组织化学研究结果在蛋白水平上初步解释了人参抑制小鼠胸腺细胞凋亡的作用机制。     

IL—2,IL—6对地塞米松诱导小鼠胸腺细胞凋亡的调节作用

目的：以Ｄｅｘ诱导小鼠胸腺细胞凋亡为模型,研究细胞因子（ＩＬ－２、ＩＬ－６）对小鼠胸腺细胞凋亡的调节作用。方法：应用ＰＩ法检测亚二倍体细胞,二苯胺法测定胸腺细胞片段化ＤＮＡ含量（％）,ＤＮＡ凝胶电泳,流式细胞计分析胸腺细胞表型、测定高钙细胞百分比。结果：发现地塞米松（Ｄｅｘ）与小鼠胸腺细胞共同培养引起细胞凋亡,胸腺细胞减少以ＣＤ４^ ＣＤ８^ 细胞最明显。细胞凋亡具有时间效应并与Ｄｅｘ浓度有关。３～４     

In vivo induction of apoptosis (programmed cell death) in mouse thymus by administration of lipopolysaccharide.

In vivo administration of bacterial lipopolysaccharide to mice induced DNA fragmentation in the thymus. Fragmented DNA was confirmed by agarose gel electrophoresis and laser flow cytometry. DNA fragmentation was predominantly detected in the thymus of young mice, while it was undetectable in the spleen, bone marrow, and lymph nodes. DNA fragmentation in the thymus was roughly dependent on the dose of lipopolysaccharide injected and reached the peak about 18 h after the injection. The addition of lipopolysaccharide to in vitro cultures of thymocytes did not cause DNA fragmentation, suggesting that lipopolysaccharide was unable to induce apoptosis of thymocytes directly. The injection of lipopolysaccharide induced no significant DNA fragmentation in adrenalectomized mice. The injection of anti-tumor necrosis factor alpha antibody together with lipopolysaccharide partially inhibited the appearance of DNA fragmentation in the thymus. On the basis of the fact that DNA fragmentation is one of the characteristics typical in apoptotic cell death, it was suggested that lipopolysaccharide could induce apoptosis in the mouse thymus in vivo. This apoptosis in the thymus might be mediated mainly by the adrenal hormones, but it is likely that tumor necrosis factor alpha might also participate in it.     

Cross-Linking the TCR Complex Induces Apoptosis in CD4+8+ Thymocytes in the Presence of Cyclosporin A

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD⁺8⁺ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.     

糖皮质激素诱导小鼠胸腺细胞死亡的形态学改变

目的 探讨糖皮质激素诱导小鼠胸腺细胞死亡的形态学改变. 方法 4～6周龄雌性BALB/C小鼠腹腔注射地塞米松,采用末端转移酶介导的dUTP缺口末端标记(TUNEL)法检测DNA单链或双链的裂解,TUNEL和酸性磷酸酶(ACP)双重染色法进行酸性磷酸酶的检测,观察细胞凋亡后吞噬细胞的活动,透射电子显微镜检测凋亡细胞和吞噬细胞的超微结构.结果 光镜观察地塞米松组,TUNEL阳性细胞较多并且分散在皮质各处,凋亡指数为0.460±0.012;正常对照组TUNEL阳性细胞数目较少,凋亡指数为0.020±0.001,与地塞米松组比较,差异有显著性(P<0.05).TUNEL和酸性磷酸酶双重染色法显示,所有TUNEL阳性胸腺细胞均被ACP阳性细胞吞噬.透射电子显微镜观察发现,大部分凋亡细胞均被吞噬细胞所吞噬,少量未被吞噬的胸腺细胞表现出大范围的染色质浓集,这些少量未被吞噬的胸腺细胞显然是死细胞. 结论 典型的细胞凋亡的特点是DNA裂解,但这不是糖皮质激素诱导胸腺细胞死亡的主要方式,死亡的胸腺细胞均是被ACP阳性细胞所吞噬;糖皮质激素对胸腺细胞的首要影响是可诱导无DNA裂解的细胞固缩.     

An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes

CAD (caspase-activated DNase) can cause DNA fragmentation in apoptotic cells. Transgenic mice that ubiquitously express a caspase-resistant form of the CAD inhibitor (ICAD) were generated. Thymocytes prepared from the mice were resistant to DNA fragmentation induced by a variety of stimuli. However, similar numbers of TUNEL-positive cells were present in adult tissues of transgenic and wild-type mice. Exposure to gamma-irradiation caused a striking increase in the number of TUNEL-positive cells in the thymus of wild-type, but not transgenic, mice. TUNEL-positive nuclei in transgenic mice were confined to thymic macrophages. When apoptotic thymocytes from the transgenic mice were cocultured with macrophages, the thymocytes underwent phagocytosis and their chromosomal DNA underwent fragmentation. This DNA fragmentation was sensitive to inhibitors that block the acidification of lysosomes. Hence, we conclude that the DNA fragmentation that occurs during apoptosis not only can result cell-autonomously from CAD activity but can also be attributed to a lysosomal acid DNase(s), most likely DNase II, after the apoptotic cells are engulfed.     

Nonylphenol-induced thymocyte apoptosis involved caspase-3 activation and mitochondrial depolarization

Although the effect of 4-nonylphenol on cells of immune system have long been recognized, little is known about the effect of 4-nonylphenol on the induction of apoptosis and related signaling events in the lymphoid cells. In the present study, we used cultured thymocytes of mice to investigate the ability of 4-nonylphenol to induce the apoptosis of thymocytes and to explore the role of signal transduction pathway leading to apoptosis. The results showed that the cytotoxic effects of 4-nonyphenol involved DNA fragmentation (DNA ladder), characteristic of apoptosis. Staining of 4-nonyphenol-treated thymocytes with DNA-binding fluorochrome Hoechst 33258 showed the typical apoptotic nuclei condensation and fragmentation of chromatin. The rates of apoptosis of the 4-nonylphenol-treated thymocytes increased significantly at 4 and 6 h, which were determined by analysis of hypodiploid cells and FITC-Annexin V and PI double staining. Flow cytometer analysis also revealed that the loss of mitochondrial membrane potential and increased activity of caspase-3 occurred concomitantly with the onset of 4-nonyphenol-induced apoptosis. Furthermore, a caspase-3 inhibitor, z-DEVD-fmk protected thymocytes from apoptosis induced by 4-nonyphenol. These results suggest that 4-nonylphenol induces thymocyte apoptosis via caspase-3 activation and mitochondrial depolarization.