人 CD4+T细胞在肠道细菌抗原刺激前后CD25与FOXP3的表达

目的：探讨新鲜分离及自身肠道菌群抗原刺激后的人外周血CD4^＋T细胞CD25和FOXP3表达的相关性及变化规律．方法：分离人的外周血单核细胞和淋巴细胞，单核细胞经自身肠道菌群抗原刺激后与淋巴细胞混合培养，新鲜分离及混合培养14d的淋巴细胞用三色流式细胞术在单细胞水平同时检测CD4，CD25和FOXP3的表达．结果：新鲜分离的淋巴细胞约43．4％表达CD4，在CD4^＋T细胞中，约5．8％表达CD25，而高表达的只有1．9％，约5．2％表达FOXP3，CD25和FOXP3均表达的占3．3％，CD25^hi FOXP3^＋细胞占1．6％，CD4^-细胞几乎不表达FOXP3．经抗原刺激后，淋巴细胞总数均轻度增加（与未刺激组比较，平均增加21．0％，P〈0．001）．CD4^＋细胞的比例均明显上升（67．1％vs43．4％，P〈0．001），CD25^＋，FOXP3^＋及CD25^＋FOXP3^＋细胞的比例变化不大．CD4^＋CD25^＋FOXP3^＋T细胞占CD4^＋CD25^＋T细胞的比例及CD4^＋CD25^hi FOXP3^＋T细胞占CD4^＋CD25^hiT细胞的比例在所有研究对象中均呈下降趋势（前者从59．8％降到52．0％，后者从86．3％降到77．9％，均P〈0．05）．结论：CD25和FOXP3的表达具有一定的相关性，但CD25及CD25^hi均不能很可靠地标记人类的调节性T细胞，尤其是在淋巴细胞受到抗原刺激以后．肠道菌群抗原作为外来抗原刺激自身的淋巴细胞后，活化增殖的细胞中主要是CD4^＋CD25^＋FOXP3^-反应性T细胞，而不是CD4^＋CD25^＋FOXP3^＋调节性T细胞．     

脐血和成人外周血CD4^＋CD25^＋调节性T细胞的分离及功能特征的比较

目的分离成人外周血、脐血CD4^＋CD25^＋调节性T细胞,并检测其功能,以了解脐血CD4^＋CD25^＋T细胞的特性。方法应用免疫磁珠分选法从健康成人外周血、脐血淋巴细胞中分离纯化CD4^＋CD25^＋、CD4＋CD25-T细胞。应用流式细胞术检测分离纯度,胎盘蓝染色检测细胞存活率;RT-PCR技术检测CD4^＋CD25^＋、CD4＋CD25-T细胞中Foxp3的mRNA的表达;体外增殖实验检测其对CD4＋CD25-T细胞的免疫抑制作用。结果 MACS分离的CD4^＋CD25^＋T细胞、CD4＋CD25-T细胞纯度达（92.7±1.6）%、（90.3±1.2）%,细胞存活率达（95.3±2.1）%;体外经抗CD3、CD28单克隆抗体刺激培养的脐血及成人外周血CD4^＋CD25^＋T细胞同时得到扩增,而且高表达Foxp3;CD4^＋CD25^＋T细胞能有效的抑制效应T细胞的增殖,当效靶比为1︰1时,其抑制效应T细胞的作用最强,成人外周血抑制率为（81.36±1.61）%、脐血抑制率为（90.74±2.43）%。结论脐血和成人外周血CD4^＋CD25^＋T细胞是一群具有免疫抑制功能的调节性T细胞,与成人外周血相比,培养后的脐血CD4^＋CD25^＋T细胞比成人外周血有更强的免疫抑制功能。     

Graves病患者外周血CD4+CD28-T淋巴细胞的检测和分选

目的 分析CD4^＋CD28ˉT淋巴细胞Graves病（毒性弥漫性甲状腺肿）患者外周血的表达及探索纯化该T细胞的有效方法。方法 运用常规密度梯度法分离Graves病患者外周血单个核细胞,用CD4、CD28单克隆抗体进行免疫荧光双标记后进行流式细胞术检测和分选。结果 Grave病患者外周血T细胞亚群中,CD4^＋CD28ˉ的表达率明显高于正常人（P〈0．01）,采用流式细胞术和单克隆抗体双标记对CD4^＋CD28ˉT淋巴细胞进行快速无菌分选是可行的。结论 流式细胞术可用于Graves病患者外周血CD4^＋CD28ˉT淋巴细胞的检测和分选。     

CD34、CD59在阵发性睡眠性血红蛋白尿中的表达及意义

目的研究阵发性睡眠性血红蛋白尿（PNH）患者骨髓CD34^＋细胞比率及外周血粒、红细胞CD59表达阳性率并探讨其临床意义。方法采用流式细胞术检测20例PNH患者骨髓单个核细胞（BMMNC）中CD34^＋细胞比率及外周血中粒、红细胞CD59表达阳性率。结果PNH患者CD34^＋细胞比率显著低于正常人（P〈0．01）;其中增生减低组CD34^＋细胞比率低于增生活跃、增生明显活跃组（P均〈0．05）,而后两组之间差异无显著性（P〉0．05）。正常人外周血粒、红细胞CD59表达阳性率均〉95％,其中粒细胞CD59表达稳定性较好;PNH患者外周血粒、红细胞CD59表达阳性率与正常对照组相比均显著减低（P〈0．01）;粒细胞CD59在不发、偶发、频发组中表达逐渐减低且差异均有显著性（P〈0．05）,红细胞CD59表达则在三组之间差异无显著性（P〉0．05）。结论CD34^＋造血干细胞减少可能参与PNH发病;外周血粒、红细胞CD59检测是诊断PNH的特异性及敏感性方法,其中粒细胞CD59缺失程度对判断病情严重与否具参考价值。     

多发性抽动症外周血中T淋巴细胞亚群CD45RA^＋、CD45RO^＋的表达及临床意义

目的观察多发性抽动症（Tourette’s syndrome，TS）患儿外周血中T淋巴细胞亚群CD4^＋CD45RA^＋、CD4^＋CD45RA^＋细胞的表达及临床意义。方法利用流式细胞术（Flow Cytometry，FCM）检测34例TS患儿外周血中T淋巴细胞亚群CD4^＋CD45RA^＋、CD4^＋CD45RA^＋细胞的表达，同期检测35名年龄、性别无差异的健康儿童为对照。结果TS组外周血中CD4^＋CD45RA^＋细胞百分率（63．70±5．57）较正常对照组（45．39±7．70）高，t=10．29，P〈0．01；TS组外周血中CD4^＋CD45RO^＋细胞百分率（17．09±4．46）较正常对照组（41．99±8．47）低，t=11．11，P〈0．01；TS组CD4^＋CD45RA^＋／CD4^＋CD45RO^＋比值（4．32±1．51）较正常对照组（1．21±0．37）高，t=7．78，P〈0．01；TS组CD4^＋CD45RA^＋、CD4^＋CD45RO^＋双阳性率（14．63±7．56）较正常对照组（9．37±3．20）高，t=2．89，P〈0．05；均有显著统计学意义。结论TS患儿存在免疫功能紊乱，主要表现为外周血中CD4^＋CD45RA^＋细胞百分率明显增高，CD4^＋CD45RO^＋细胞百分率明显降低，TS患儿CD4^＋CD45RA^＋／CD4^＋CD45RO^＋失衡，TS患儿CD4^＋CD45RA^＋、CD4^＋CD45RO^＋双阳性率较高，这可能在TS发病机制中起着重要作用。     

肾癌患者多电极射频消融治疗前后细胞免疫功能的研究

目的研究肾癌患者接受射频消融治疗前后机体T淋巴细胞及红细胞免疫功能的变化。方法分析29例肾细胞癌患者射频消融治疗前和治疗后7、15和30d外周血淋巴细胞亚群（CD3^＋、CD4^＋、CD8^＋、CD4^＋/CD8^＋）和红细胞C3b受体花环（RBC-C3bR）及免疫复合物花环（RBC-ICR）形成率。结果所有肾癌患者在射频消融治疗后7d,CD3^＋,CD4^＋,CD4^＋/CD8^＋较治疗前明显升高（P〈0.05）;15d后RBC-C3bR形成率明显高于治疗前,而RBC-ICR形成率低于治疗前（P〈0.05）。结论多电极射频消融治疗肾癌,可以改善患者的细胞免疫状态。     

中国恒河猴CD8+T细胞缺失模型制备

目的：测试用抗-HuCD8^＋T细胞单克隆抗体方法去除中国恒河猴体内CD8^＋T细胞效果，为建立CD8^＋T细胞缺失恒河猴模型提供实验依据。方法：抗-HuCD8^＋T抗体cM-T807在0、3、7d注射2只中国恒河猴，不同的时间取外周血，腹股沟和腋下淋巴结。制备CD3／CD4／CD8标记的外周血和淋巴细胞悬液，流式法测定恒河猴外周血和淋巴结中CD8^＋T细胞的去除效果。结果：注射了cM-T807的中国恒河猴，24h后，外周血中的CD8^＋T细胞缺失99．9％，持续27d；168h后，淋巴结中CD8^＋T细胞缺失约90．0％，持续21d。结论：首次证实抗-HuCD8^＋T细胞抗体cM-T807能有效去除中国恒河猴体内CD8^＋T细胞，为建立CD8^＋T细胞缺失动物模型（CD8^＋T Cells del）奠定基础。     

恶性淋巴瘤患者外周血T细胞CD8~+CD28~-表达及意义

目的探讨恶性淋巴瘤患者外周血T细胞CD8^＋CD28^-表达及意义。方法采用流式细胞仪检测35例恶性淋巴瘤患者外周血T细胞CD8^＋CD28^＋及CD8^＋CD28^-。结果恶性淋巴瘤患者外周血T细胞CD8^＋CD28^＋及CD8^＋CD28^-与正常对照有显著性差异（P〈0．05）：治疗有效后两者趋于正常（P〉0．05）；而无效组与正常对照组相比仍有显著性差异（P〈0．05）。结论恶性淋巴瘤患者外周血T细胞CD8^＋CD28^＋及CD8^＋CD28^-表达异常，不同治疗效果表达不同，恶性淋巴瘤患者有免疫功能异常，治疗有效后免疫功能恢复正常。     

中国HCV/HIV合并感染者或HIV单一感染者T淋巴细胞表面CD25和CXCR3表达的研究

目的探讨T淋巴细胞表面CD25分子和趋化因子受体CXCR3，在丙肝病毒（HCV）单一感染，艾滋病病毒（HIV）单一感染和HCV／HIV合并感染过程中的表达及意义。方法分离HCV感染组（n=21），HIV感染组（n=14），HCV／HIV感染组（n=28）及正常对照组（n=30）人外周血单个核细胞。采用流式细胞术。计数CD4^＋和CD8^＋t淋巴细胞数，检测外周血CD4^＋CD25^＋细胞百分含量和CD4^＋和CD8^＋淋巴细胞表面CXCR3的表达水平。结果CD4^＋T细胞表面CD25的表达在HCV感染组轻度升高，而在HIV感染组及HCVIHIV合并感染组CD25的表达显著降低（P〈0．001）。HIV感染组及HCV／HIV合并感染组CD4^＋T细胞表面CXCR3表达显著降低（P〈0．001）。CD8^＋T细胞表面CXCR3表达显著升高（P〈0．001）；HCV感染组CD4^＋及CD8^＋T细胞表面CXCR3表达轻度升高，但差异不显著。结论结果提示中国病毒感染者外周血T淋巴细胞表面CD25和CXCR3分子表达水平和机体免疫调节功能受到HIV感染的影响。     

乙型肝炎患者肝组织CD8+T淋巴细胞CD28的表达及临床意义

目的：探讨乙型肝炎患者肝组织CD8＋T淋巴细胞CD28的表达及其临床意义。方法：将100例乙型肝炎患者按照肝组织病理变化分为轻、中、重度,15名健康人肝组织为对照组,采用免疫组织化学双标记法检测肝组织CD8＋CD28＋T细胞和CD8＋CD28-T细胞。结果：乙型肝炎患者肝组织CD8＋CD28＋T细胞和CD8＋CD28-T细胞水平明显高于对照组（P〈0.05）。轻度乙型肝炎患者肝组织内CD8＋CD28＋T细胞水平较对照组低,乙型肝炎中度和重度患者CD8＋CD28＋T细胞和CD8＋CD28-T细胞明显高于对照组（P〈0.01）,且CD8＋CD28＋T细胞随着炎症分级逐级升高,CD8＋CD28-T细胞却随着炎症分级逐级减低。结论：肝组织内CD8＋T淋巴细胞表面CD28的表达强度与肝组织病变程度密切相关;肝组织内CD8＋CD28＋T细胞水平可作为判断乙型肝炎患者肝组织损伤的一个免疫学指标;肝组织内CD8＋CD28-T细胞可能是乙型肝炎持续感染的主要原因。     

Decreased expression of complement regulatory proteins, CD55 and CD59, on peripheral blood leucocytes in patients with type 2 diabetes and macrovascular diseases

Background Macro- and microvascular diseases are the leading cause of morbidity and mortality in diabetic patients, but their mechanisms remain unclear. Recent reports provide evidence that the levels of CD55 and CD59 are decreased in diabetic microvascular diseases. However, very little is known about the levels of CD55 and CD59, the relationship between them and carotid artery intima-media thickness, and the effects of statins on CD55 and CD59 in diabetic macrovascular diseases. Methods The mean fluorescence intensity （MFI） of CD55 and CD59 expression on peripheral blood leucocyte subsets （lymphocytes, monocytes and neutrophils） was studied using flow cytometry, and carotid artery intima-media thickness was measured using B-mode ultrasonography in 23 healthy subjects （controls）, 19 patients with type 2 diabetes （T2DM）, and 43 patients with type 2 diabetes and macrovascular diseases （T2DM-M）. The patients with T2DM-M were assigned to two subgroups based on whether statins were used： group with statins （n=-23） and group without statins （n=20）. Results Compared with the controls and T2DM, the MFI of CD55 positive neutrophils was significantly lower in T2DM-M （P=0.049 vs controls and P=0.033 vs T2DM）; similarly, the MFI of CD59 positive monocytes was also lower in T2DM-M （P=0.038 vs controls and P=0.043 vs T2DM）. The MFI of CD59 positive neutrophils in T2DM-M was lower than in T2DM （P=0.032）. The levels of CD55 and CD59 were negatively associated with age and blood pressure （r=-0.245- -0.352, P=0.041-0.003）, but not acute-phase reactants and carotid artery intima-media thickness. The levels of CD55 and CD59 increased after treatment with statins, but the results were not significantly different （P 〉0.05 ）. Conclusions CD55 and CD59 expressions on peripheral blood leucocytes are decreased in T2DM patients with macrovascular diseases. The results suggest that the decreased levels of complement regulatory proteins might play an important role in diabetic macrovascular diseases.     

强直性脊柱炎患者外周血单核及T淋巴细胞CD147的表达及意义

目的 观察强直性脊柱炎(AS)患者外周血单核及T淋巴细胞中CD147的表达情况,初步探讨CD147在AS发病及疾病活动中的意义.方法 入组AS患者、类风湿关节炎(RA)患者及健康对照者(HC)各30例,以流式细胞术分析比较各组间外周血单核及T淋巴细胞CD147表达的差异,并经逆转录-聚合酶链反应(RT-PCR)法检测比较其外周血单个核细胞(PBMC)CD147 mRNA表达的差异;进而将AS患者单核及T淋巴细胞CD147表达水平与常用的疾病活动性指标行相关分析.结果 AS、RA及HC组外周血单核细胞CD147的平均荧光强度(MF1)分别为213.5±37.8、228.7±49.7及163.6±44.8,T淋巴细胞CD147的MFI则分别为36.8±10.1、40.2±10.5及28.3±10.6,AS组单核及T淋巴细胞CD147的表达水平均略低于RA患者,但差异均无统计学意义(均P＞0.05),而AS与RA组单核及T淋巴细胞CD147的表达水平均高于HC组,差异均有统计学意义(均P＜0.05);AS与RA组PBMC中CD147 mRNA表达水平均高于HC组(P＜0.05),而AS与RA组间差异无统计学意义(P＞0.05);AS患者单核及T淋巴细胞CD147 MFI与其ESR及CRP均存在正相关(P＜0.05).结论 AS患者单核及T淋巴细胞中CD147的表达较正常升高,其水平与患者ESR及CRP存在正相关,提示CD147可能是AS患者疾病发生发展的重要影响因素.     

Reduction of CD55 and/or CD59 in red blood cells of patients with HIV infection.

BACKGROUND: Anemia is a common feature in HIV infection. An increased sensitivity of lymphocytes from HIV patients to lysis by complement has been correlated with a decreased expression of CD55 and CD59 in their surface. The aim of this study was to evaluate CD55/CD59 presence in red cells of HIV patients and explore possible correlations with clinical parameters. MATERIAL/METHODS: CD55/CD59 expression was evaluated in erythrocytes of 37 patients (30M/7F, median age: 39 years) with HIV infection (25 also having hemophilia), 121 controls, and 8 PNH patients using the sephacryl-gel microtyping system. Ham and sucrose tests were also performed. RESULTS: Anemia was present in 14/37 (37%) HIV patients. Interestingly, all HIV patients had deficient CD55 and/or CD59 erythrocytes: 8 (21%) for both CD55 andCD59 and 29 (78%) isolated CD55 and/or CD59 negativity. Deficient erythrocytes did not account for more than 10% of the total in the vast majority of patients. In controls, only 2 (1%) had red cells with double CD55/CD59 negativity and 3 (2%) had isolated deficiency. All PNH patients had a simultaneous CD55/CD59 deficiency. Positive Ham and sucrose tests were found only in PNH. There was no correlation between the presence of deficient CD55/CD59 erythrocytes and anemia, hemolysis, antiretroviral therapy, CD4+ counts, viral load, or concomitant hepatitis C infection in HIV patients. CONCLUSIONS: This study provides evidence supporting the presence of erythrocytes with CD55 and/or CD59 deficiency in HIV. Further studies using molecular techniques will be required to clarify the exact role of this deficiency in HIV patients.     

Phenotypic analysis of affected peripheral erythroid for CD59 in paroxysmal nocturnal hemoglobinuria

Objective To determine whether affected reticulocytes could be a reliable marker for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), we analyzed CD59-antigen expression on the membranes of reticulocytes and erythrocytesMethods We studied 10 PNH patients and 5 healthy volunteers by two-color flow cytometry with a membrane permeable fluorescent dye, thiazole orange (TO), and anti-CD59 monoclonal antibodies (MoAb) TO was introduced to gate reticulocytes and anti-CD59 MoAb were used to identify glycosylphosphatidylinositol (GPI)-deficient cellsResults Cells from healthy individuals were only CD59 positive However, in all PNH patients, CD59-antigen expression could be divided into 3 types: type Ⅰ cells (CD59 normally positive), type Ⅱ cells (CD59 partly positive) and type Ⅲ cells (CD59 negative) The majority of reticulocytes belonged to type Ⅲ cells, GPI-deficient cells (610%) In addition, the percentage of affected reticulocytes was higher than that of erythrocytesConclusions Analyzing PNH reticulocytes was important, because most patients had elevated numbers of reticulocytes, which represent more closely the recent erythroid output of BM However, circulating mature erythrocytes were subject to complement- mediated intravascular lysis Therefore, the percentage of abnormal erythrocytes may not accurately reflect the proliferation rate of normal and abnormal erythroid progenitor cells Thus, affected reticulocytes could be a more reliable indicator for the diagnosis of PNH than mature erythrocytes.     

CD55,CD59抗原表达在PNH合并全血细胞减少症诊断中的应用

目的 探讨红细胞和粒细胞膜CD55与CD59抗原表达在阵发性睡眠性血红蛋白尿症(PNH)合并全血细胞减少症(PCP)诊断中的应用.方法采用流式细胞术结合直接免疫荧光法,检测PCP患者病因可疑为PNH的的外周血红细胞和粒细胞膜 CD55与CD59抗原的表达.结果 125例中,PNH32例,非PNH 93例(包括再生障碍性...     

CD59特异位点短肽封条对T细胞封闭作用

目的 探讨含有CD59特异位点的短肽封条对T细胞的封闭作用及其可能机制.方法 建立HeLa肿瘤小鼠模型,眼球取血分离血清测定补体C3、C4,分离培养鼠胸腺T淋巴细胞、分别加入CD59-mAb、CD59特异位点短肽封条作用48 h后,MTT法检测T细胞增殖情况.结果 肿瘤小鼠补体C3、C4含量明显降低(t=30.970、16.789,P<0.01);CD59-mAb对T细胞增殖起促进作用,而含有CD59特异位点的短肽封条能抑制T细胞增殖,两者的作用与正常对照相比差异有显著性(t=5.428、4.726,P<0.01).结论 CD59参与T细胞信号转导而与补体抑制作用无关,含有CD59特异位点的短肽封条对T细胞起封闭作用.     

经鼻腔激光疗法对脑梗死病人外周血淋巴细胞CD抗原的影响

目的探讨经鼻腔激光照射疗法对脑梗死病人治疗前后外周血CD抗原的影响.方法用T细胞检测试剂盒进行测定.结果治疗前脑梗死病人外周血T淋巴细胞CD3、CD8占的比例下降,而CD4/CD8上升,治疗后CD3、CD8比例上升,CD4/CD8比例下降.结论经鼻腔激光照射疗法可改善CD抗原比例,对脑梗死的治疗有一定的临床意义.     

CD59与CD55锚固蛋白对T细胞活化信号转导的作用

目的研究糖基磷脂酰肌醇(GPI)锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的作用。方法应用siRNA技术,构建针对CD55与CD59基因的重组载体pSUPER-siCD55、pSUPER-siCD59。实验分为未转染的Jurkat细胞组(Ⅰ组)、转染pSUPER-siCD59重组质粒的Jurkat细胞组(Ⅱ组)及转染pSUPER-siCD55重组质粒的Jurkat细胞组(Ⅲ组)。RT-PCR方法检测转染细胞中CD55和CD59基因mRNA的表达。噻唑蓝(MTT)比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对3组Jurkat细胞的增殖效应以及细胞内钙离子的变化。结果与Ⅰ组比较,Ⅱ组细胞CD59和Ⅲ组细胞CD55 mRNA的表达均明显减少(F=595.14、699.06,q=45.70~47.25,P<0.05)。Ⅰ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅱ组、Ⅲ组(F=97.02、189.47,q=13.69~26.39,P<0.01),Ⅱ组低于Ⅲ组(q=5.43、6.42,P<0.05)。结论 CD59和CD55在T细胞活化信号转导通路中存在协同效应,其中CD59起到重要作用。