姜黄素对CD34~+人急性髓系白血病细胞增殖抑制作用及机制探讨

目的:探讨姜黄素对CD34+人急性髓系白血病细胞株KG1a和Kasumi-1增殖抑制作用及其机制。方法:以不同浓度姜黄素(0、20、40、80μmol/L)处理KG1a和Kasumi-1细胞48 h,采用台盼蓝染色计数法检测细胞活率,Western blot法测定姜黄素对细胞NF-κB P65核蛋白表达影响,免疫荧光检测姜黄素对细胞NF-κB P65核蛋白移位影响。结果:姜黄素可显著抑制KG1a和Kasumi-1细胞生长,随着姜黄素浓度的增加,细胞数量逐渐下降。姜黄素可下调KG1a和Kasumi-1细胞P65核蛋白表达,抑制NF-κB P65核移位,使NF-κB处于失活状态。结论:姜黄素可抑制KG1a和Kasumi-1细胞增殖,其机制可能与姜黄素抑制NF-κB P65核蛋白表达有关。     

急性髓系白血病细胞血管细胞黏附分子1、CD34和CD117的表达

背景:血管细胞黏附分子1与白血病浸润密切相关,白血病细胞本身是否表达血管细胞黏附分子1,以及与疾病难治是否相关尚无定论。目的:分析血管细胞黏附分子1、CD34、CD117在急性髓系白血病细胞表面的表达,3者之间的相互关系及与难治性急性髓系白血病的相关性。方法:采用流式细胞技术检测16例急性髓系白血病细胞中血管细胞黏附分子1、CD34、CD117的表达,其中难治组6例,非难治组10例;同时以正常骨髓单个核细胞标本作对照。结果与结论:急性髓系白血病细胞CD34、CD117表达高于对照组(P<0.05)。难治组急性髓系白血病细胞CD34表达明显高于非难治组(P<0.05)。难治组与非难治组CD117表达差异无显著性意义(P>0.05)。急性髓系白血病细胞血管细胞黏附分子1表达与对照组比较差异无显著性意义(P>0.05)。难治组与非难治组血管细胞黏附分子1表达差异无显著性意义(P>0.05)。表明急性髓系白血病细胞伴CD34表达,为不良预后指标之一,CD117、血管细胞黏附分子1表达与其是否难治无明显相关性。     

三氧化二砷联合姜黄素对KG1a细胞的协同杀伤作用

本研究旨在探讨三氧化二砷联合姜黄素对KG1a细胞的增殖与凋亡的影响及可能的机制.采用MTT法检测细胞存活率;甲基纤维素集落形成实验检测细胞成集落能力;流式细胞术检测细胞表面分子、细胞凋亡率及细胞周期变化;瑞氏姬姆沙染色法观察细胞形态;Westerrn blot检测细胞BCL-2、BAX、PARP蛋白表达.结果表明：KG1a细胞表型为CD34＋ CD38-,HL-60细胞表型为CD34＋ CD38＋;前者成集落能力比后者强.姜黄素及三氧化二砷单用对KG1a细胞增值抑制作用均具有剂量依赖性.联合用药与单药对比,前者细胞存活率、克隆形成集落数更低,而细胞凋亡率更高.联合用药能够降低细胞的BCL-2、PARP两种蛋白表达、增加BAX蛋白表达.结论：KG1a细胞是比HL-60细胞更早期的白血病干/祖细胞.三氧化二砷联合姜黄素能更有效抑制KG1a细胞增殖及诱导其凋亡,其机制可能与BCL-2、PARP蛋白表达下调、BAX蛋白表达上调有关.     

山奈素对KG1a细胞增殖的抑制作用及机制研究

目的：探讨山奈素对急性髓系白血病(AML)KG1a细胞增殖抑制作用及其相关作用机制。方法：取对数生长期的人AML KG1a细胞，培养液中分别加入山奈素药液0、25、50、75、100μg/ml，溶剂对照组加入二甲基亚砜，干预24和48 h后，采用CCK-8法检测细胞增殖率；另设白细胞介素-6(IL-6)与山奈素联用组(加20μg/L的IL-6和75μg/ml的山奈素)，给药培养48 h后，流式细胞术检测KG1a细胞周期及凋亡情况，线粒体膜电位(MMP)检测试剂盒(JC-1法)检测KG1a细胞MMP，蛋白免疫印迹法检测KG1a细胞JAK2/STAT3通路相关蛋白表达。结果：山奈素25、50、75、100μg/ml组细胞增殖率明显降低(P<0.05)，且随着山奈素剂量的增大，细胞增殖率逐渐降低(r_{24 h}=-0.990、r_{48 h}=-0.999)(P<0.05);75μg/ml的山奈素干预48 h后对细胞增殖的抑制作用已达到半数有效剂量；与空白对照组相比，山奈素25、50、75μg/ml组细胞在G₀/G_...     

初诊急性髓系白血病骨髓CD34+CD38-细胞群比例是初次诱导缓解率的预后因素

为了研究非M3急性髓系白血病(AML non-M3) CD34+ CD38 -细胞群及其G0期比例与临床和实验室特征的关系,使用流式细胞仪检测40例初治AML non-M3骨髓单个核细胞CD34和CD38的表达,测定各群细胞的细胞周期,并分析CD34+ CD38 -细胞群及其G0期比例与临床实验室特征及初次诱导缓解率的关系.结果显示,CD34+ CD38 -细胞群及其G0期的比例与染色体核型、初诊WBC计数及FLT3/ITD阳性均无明显相关性,但与诱导治疗后骨髓中幼稚细胞比例相关.诱导停疗第7天骨髓中有幼稚细胞患者CD34+ CD38-细胞比例为(12.47±26.26)％,诱导停疗第7天无幼稚细胞患者CD34+ CD38 -细胞比例为(2.62±7.20)％ (p =0.031).诱导停疗第1天骨髓中可见幼稚细胞患者CD34+细胞群比例为(17.40±21.20)％,而诱导停疗第1天无幼稚细胞患者该比例为(5.64±6.96)％(p=0.00l).CR组患者治疗前CD34+ CD38 -细胞群的比例为(2.51±9.72)％,明显低于非CR组患者(24.92 +27.04％) (p =0.001).而在AMI non-M2b患者中,CR组患者治疗前CD34+ CD38 -细胞群的比例为(1.60±4.82)％,较非CR组患者更为降低(p ＜0.001).单因素分析显示,诱导化疗后是否取得CR与年龄(p=0.022)、CD34+ CD38 -细胞群比例(p=0.008)、诱导停疗第7天骨髓幼稚细胞比例(p=0.011)相关.多因素分析显示,仅有CD34+ CD38 -细胞群的比例与是否获得CR有相关趋势(p =0.052).结论:初治AML患者CD34+CD38 -细胞群的比例是AML初次诱导缓解率的预后因素.     

CD34的分子生物学研究进展

Civin等和Tindle等先后在1984年和1985年用人髓系细胞系KG—1a和KG—1制备了单克隆抗体(单抗)My10和BI.3C5。这两种单抗能与大多数骨髓来源的干、祖细胞,急性髓系和淋巴系白血病不成熟细胞以及血管内皮细胞上所表达的一种105—120kD膜蛋白特异结合。这种膜蛋白的表达与细胞分化阶段有关,而与系别(lineage)无关,1986年第三届国际人类白细胞分化抗原协作组会议将其命名为CD34。通过CD34对造血细胞进行分选及对CD34~ 细胞的性能研究,已获可喜成果。然而CD34在有关细胞增殖、分化过程中起何作用,迄今     

抗-CD36单克隆抗体对人CD34~+造血干(祖)细胞增殖和分化的影响

目的探讨抗-CD36单克隆抗体对人CD34~+造血干(祖)细胞体外增殖和分化的影响。方法选择无各种产科并发症的健康足月产妇3名,取脐带血20 mL/(人)份,以Ficoll细胞分离液(1.077 g/mL)密度梯度800 g离心30 min后,流式细胞仪分选CD34~+造血干(祖)细胞,培养2—3代,四唑盐(MTT)比色法检测抗-CD36单克隆抗体对CD34~+细胞生长的影响;流式细胞术检测细胞凋亡,Annexin V/PI双染法和碘化丙啶(PI)单染法检测细胞周期。观察抗-CD36单克隆抗体对CD34~+造血干(祖)细胞凋亡和细胞周期的影响、对造血干(祖)细胞红系分化能力以及对红系集落形成单位(CFU-E)和红系爆式集落形成单位(BFU-E)生成的影响。结果流式细胞仪分选出经Ficoll分离脐带血获得的单个核细胞中约0.44%CD34~+造血干(祖)细胞。2、8、32、64、128μg/mL抗-CD36单克隆抗体分别与CD34~+造血干(祖)细胞体外共培养:单纯CD34~+造血干(祖)细胞培养(正常)组、抗-CD36单克隆抗体2及32μg/mL组,IgG(对照)组的OD值分别为1.05±0.12 vs 0.9±0.15 vs 0.81±0.11 vs 1.06±0.18(P0.01)。Annexin V流式检测凋亡率(%):正常组、对照组与2μg/mL抗-CD36单克隆抗体组分别为10.13SymbolqB@1.42 vs 10.51SymbolqB@1.75 vs 24.57SymbolqB@2.75(P0.05)。G_1/S值:正常组、对照组与2μL/mL抗-CD36单克隆抗体组分别为3.95±0.25 vs 4.36±0.55 vs 7.35±0.65(P0.05)。CD34~+造血干(祖)细胞定向分化红系(CFU-E/BFU-E克隆形成数):正常组、对照组与抗-CD36单克隆抗体组分别为169±12、172±12和85±6(P0.05)。结论抗-CD36单克隆抗体诱导人CD34~+造血干(祖)细胞凋亡,细胞增殖减少和红系CFU-E/BFU-E克隆能力降低。     

N-cadherin在维持白血病干细胞特征中的作用

自我更新和抗药性是白血病干细胞的重要特征,为阐明N-cadherin参与维持白血病干细胞特征的分子机制,本研究以CD34病细胞系KG1a为研究模型,确定N-cadherin在维持白血病细胞自我更新和抗药性中的作用。通过流式细胞仪分选出N-cadherin阳性及N-cadherin阴性两群细胞,采用集落培养及化疗药物VP16分别处理两群细胞,比较二者的体外增殖及自我更新能力,以及VP16作用于两群细胞的半数抑制浓度IC50。结果显示:N-cadherin阳性的KG1a细胞的集落形成能力显著高于N-cadherin阴性细胞。VP16作用于N-cadherin阳性细胞的IC50(220μmol/L)显著高于N-cadherin阴性细胞(151μmol/L)(p=0.04);N-cadherin介导的黏附参与了N-cadherin阳性细胞的抗药性。结论:N-cadherin在维持白血病干细胞自我更新和抗药性的特征中起重要作用。     

Aurora激酶抑制剂VX-680对慢性髓系白血病细胞增殖及凋亡的影响

本研究探讨Aurora激酶抑制剂VX-680在体外对白血病细胞系K562、KCL22和慢性髓系白血病（CML）患者骨髓CD34^＋细胞的增殖和凋亡的影响。利用CCK-8法观察VX-680对K562和KCL22细胞的增殖作用,细胞计数方法测定VX-680对CML的CD34^＋细胞增殖影响,Annexin V-PI凋亡检测试剂盒、流式细胞术分析VX-680对K562、KCL22细胞凋亡效应,集落形成试验检测VX-680对CML及正常供者（donor）的骨髓CD34^＋细胞集落形成能力影响。结果表明,Aurora激酶抑制剂VX-680（20-100 nmol/L）在处理细胞第3天时能明显抑制K562和KCL22细胞增殖（P〈0.01）,并能抑制CML患者骨髓CD34^＋细胞增殖,而且随着剂量增加,抑制作用增强;VX-680（20nmol/L）作用K562和KCL22细胞3天时可诱导细胞凋亡（P〈0.01）;集落形成试验表明,CML患者骨髓CD34^＋细胞在VX-680的体外处理下集落形成能力较正常骨髓CD34^＋细胞明显下降（P〈0.01）。结论：Aurora激酶抑制剂VX-680在体外对慢性髓系白血病细胞有抑制增殖和促进凋亡的作用。     

无血清培养体系对脐带血CD34~+细胞定向红系分化影响的比较

目的:通过3种无血清红系定向诱导分化培养体系比较,优化培养体系诱导脐带血干/祖细胞(HSPC)体外红系定向分化,以满足基础研究与临床应用。方法:应用磁珠分选脐带血单个核细胞中的CD34~+细胞;分别接种到3种培养体系(1、2、3)中并采用3阶段培养法诱导CD34~+细胞红系定向分化,在分化不同阶段进行细胞计数,瑞氏吉姆萨染色,应用流式细胞术检测细胞表面CD71、CD235a的表达,集落形成能力检测鉴定红系分化的进程。结果:体系2促HSPC增殖能力最强,体系3促红系分化效果最佳。体系2培养的细胞增殖能力均明显高于体系1、2(P 0.05);FACS分析显示,红系表面分子CD71、CD235a在体系3培养的细胞表面表达最高,分化d 15 CD235a+百分率高达(92.23±3.89)%,体系2为(84.67±3.12)%,体系1为(72.17±6.83)%(P 0.05);细胞形态学染色显示,体系3培养的细胞在分化d 12的晚幼红细胞比例为(67.67±2.08)%,是体系2的10.69倍、体系1的25.34倍(P 0.05);造血集落形成实验发现在体系3中d 3-9形成的BFU-E比例逐渐升高(r=0.99, P 0.05),体系3中BFU-E形成比率明显高于体系1、2(P 0.05)。结论:通过比较3种培养体系,筛选出体系3是促进CD34~+细胞体外红系分化最有效的体系,体系2是促增殖最有效的体系。本研究为进一步提高HSPC体外红系增殖与诱导效率奠定了技术基础,也为研究红系分化调控机制提供了体外培养体系。     

CD137 enhances cytotoxicity of CD3CD56 cells and their capacities to induce CD4 Th1 responses

CD137 (4-1BB) is a TNFR superfamily member that mediates the costimulatory signal resulting in T cells and NK cells proliferation and cytokines production, but the effects of CD137 signaling on CD3⁺CD56⁺ cell subpopulation have not been well-documented. The aim of this study was to investigate the effects of CD137 signaling on regulation of CD3⁺CD56⁺ cell function. Anti-CD137 mAb or mouse IgG1 isotype control was added to CIK cell culture to determine the effects of proliferation and anti-tumor effects on CD3⁺CD56⁺ cells. We observed that anti-CD137 mAb could dramatically promote proliferation of CIK cells. And CD137–CIK cells and CD3⁺CD56⁺ cell subpopulation within them possessed higher ability to kill tumor cell line A549. The SCID mice engrafted with A549 cells and treated with CD137–CIK cells have prolonged survival. Further studies revealed that the percentages of CD3⁺CD56⁺ cells were elevated significantly in CD137–CIK cells. The expression of NKG2D was up-regulated on CD3⁺CD56⁺ cells from CD137–CIK cells. The expression of IFN-γ, IL-2 and TNF-α increased significantly whereas the production of TGF-β₁, IL-4 and IL-10 decreased in CD3⁺CD56⁺ cells from CD137–CIK cells. In addition, anti-CD137 mAb can elevate the capacity of CD3⁺CD56⁺ cells to induce CD4⁺ Th1 responses. We further showed that the anti-CD137 mAb also had the same effects on CD3⁺CD56⁺ cells expanded from the PBMCs of patients with NSCLC. We concluded that CD137 signaling could enhance the abilities of CIK cells to kill tumor cells in vitro and in vivo via increasing the proportion of CD3⁺CD56⁺ cells and their cytotoxicity. Furthermore, CD137 signaling can elevate the capacity of CD3⁺CD56⁺ cells to induce CD4⁺ Th1 responses which may enhance their anti-tumor activity indirectly. Taken together, our studies could be considered as valuable in CIK cells-based cancer immunotherapy.     

Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.     

Differential increase of CD34, KDR/CD34, CD133/CD34 and CD117/CD34 positive cells in peripheral blood of patients with acute myocardial infarction

Objective Circulating progenitor cells (CPC) may contribute to cardiac regeneration and neovascularization after acute myocardial infarction (AMI). For potential therapeutic use, understanding the endogenous mechanisms after ischemia is inevitable. We investigated the absolute number, but also the subset composition of CD34+ CPC after AMI. Methods CD34+, KDR+/ CD34+, CD133+/CD34+ and CD117+/CD34+ CPC were analyzed by FACS in peripheral blood of 10 patients with acute MI (59±5 yrs, m/f=8/2) at day of AMI (day 0) and days 1–5. For comparison patients with stable coronary artery disease (CAD, n=12, 66±2 yrs, m/f=10/2) and young healthy volunteers (n=7, 26±2 yrs, m/f=3/4) were studied. Results CD34 and KDR/CD34, CD133/CD34, CD117/CD34 were increased day 3 and 4 after AMI. KDR+ fraction within CD34+ population remained unchanged (58.3±7.8% vs 55.3±10.6%), whereas CD133+ (64.9±3.1% vs 43.5±5.9%, P=0.006) and CD117+ fractions (71.7±5.6% vs 50.1±5.5%, P=0.02) were elevated. In CAD, all CPC and fractions were similar as AMI day 0. Healthy volunteers had more CD34+ than CAD and AMI day 0. Double positive CPC were also higher, but fractions were unchanged vs CAD with more KDR/CD34 in trend (72.8±10.6% vs 50.5±5.6%, P=0.058). After AMI both absolute numbers of CD34+ and their subset composition change, suggesting selective mobilization of CPC. Increased CPC after AMI never reach numbers of young healthy volunteers.     

CD4+CD25+和CD8+调节性T细胞的作用机制

调节性T细胞（Treg）主要在机体免疫系统中发挥负向调节作用,既能抑制不恰当的免疫反应,又能限定免疫应答的范围、程度及作用时间,对CD4^＋和CD8^＋效应性T淋巴细胞的增殖起抑制作用,因此在移植物抗宿主病、自身免疫病、过敏性疾病等的发病机制和临床治疗中有潜在的应用价值.本文重点介绍CD4^＋CD25^＋Treg和CD8^＋Treg的作用机制,并简述调节性T细胞研究面临的挑战与展望.     

Human CD3+ T lymphocytes that express neither CD4 nor CD8 antigens

CD3+ T lymphocytes expressing neither CD4 nor CD8 antigens exist in normal human peripheral blood in low frequency (approximately 3% of lymphocytes). The CD3+,4-,8- phenotype was stably maintained after in vitro culture in IL-2. Culture of CD3+,4-,8- cells in only rIL-2 generated cytotoxic T cells that lysed NK-sensitive and NK-insensitive tumor cell targets without MHC restriction. These experiments clearly show that phenotypically and functionally competent T cells expressing neither CD4 nor CD8 are present in normal peripheral blood.     

CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.     

Contribution of CD4+, CD8+CD28+, and CD8+CD28- T cells to CD3+ lymphocyte homeostasis during the natural course of HIV-1 infection.

The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.