The regulation of inhibitory receptor LAIR-1 on the differentiation of megakaryocytic lineage

目的 研究抑制性受体LAIR-1在巨核谱系细胞的表达及其对巨核细胞分化的调节作用.方法 采用流式细胞术和激光共聚焦显微镜检测LAIR-1在巨核谱系细胞的表达;免疫磁珠法纯化脐血CD34+细胞,体外无血清培养系统诱导CD34+细胞向巨核细胞分化,观察LAIR-1被抗体或配体交联激活后对巨核细胞分化的影响.结果 LAIR-1表达于人骨髓CD34+ CD41a+和CD41a+ CD42b+细胞以及脐血CD34+ CD41a -和CD34+ CD41a+细胞;脐血CD34+细胞向巨核细胞分化过程中,LAIR-1表达在倍性为2N和4N的不成熟CD41a+细胞上;交联激活LAIR-1分子能够抑制脐血CD34+细胞向巨核细胞的分化.结论 LAIR-1可能是参与巨核细胞早期分化发育的一种新的负调控分子.     

MKL1在巨核细胞分化中的表达及其作用的实验研究

目的了解MKL1在巨核细胞分化、成熟中的作用。方法以人外周血来源CD34~+细胞巨核细胞分化为模型,采用荧光实时定量PCR法研究MKL1基因在不同分化阶段巨核细胞中的表达情况,通过构建慢病毒载体在CD34~+细胞中过表达MKL1基因,利用血细胞涂片和流式细胞仪考察MKL1过表达细胞经细胞因子刺激分化后的形态、CD41~+百分比及DNA含量。结果经qPCR研究发现,MKL1基因在成熟的巨核细胞中的表达6.8倍高于未分化的CD34~+细胞,以及2倍高于单个核巨核细胞。MKL1过表达组经诱导分化后CD41~+巨核细胞百分比与多倍体细胞百分比分别为61.5%和52.9%,均显著高于对照组36.3%和33.4%的比例。结论MKL1基因在巨核细胞分化中表达逐渐上调,其外源表达促进人外周血来源CD34~+细胞巨核细胞分化和多倍体化。     

人核迁移蛋C刺激人造血干细胞增殖分化为巨核细胞研究

目的:研究人核迁移蛋C(hNUDC)促进人脐血来源的CD34 细胞增殖、分化为巨核细胞的作用.方法:使用Dynal CD34体外分离系统收集人脐血CD34 细胞, 无血清甲基纤维素半固体法体外培养CD34 细胞12 d后, 显微镜下观察hNUDC对CD34 细胞分化增殖为小、中、大巨核细胞集落的形态、数目的影响;无血清液体培养体系培养CD34 细胞10 d后, 流式细胞术检测hNUDC对CD34 细胞分化增殖为CD41 细胞的表达率及其DNA倍性的影响.结果:hNUDC能够明显促进CD34 细胞分化增殖形成中小型CFU-MK集落, 可显著增加巨核细胞表面标志物CD41 的表达, CD41 细胞中DNA倍性显著地高于血小板生成素.结论:hNUDC对促进造血干细胞增殖和分化为巨核细胞具有重要作用.     

脐带血巨核祖细胞的体外扩增

目的联合血小板生成素(TPO)、白细胞介素-11(IL-11)和肝素用脐带血CD34 细胞定向扩增巨核祖细胞。方法采用免疫磁珠法(MACS)分选CD34 细胞,用TPO、IL-11和肝素定向扩增巨核祖细胞,巨核祖细胞集落分析(CFU-MK)测定巨核祖细胞扩增倍数,流式细胞术检测巨核祖细胞分化过程中不同细胞组群(CD34 、CD41a 、CD61 、CD34 CD41a 和CD41a CD61 )的变化,免疫组织化学染色(CD41a)和透射电镜观察巨核细胞形态及趟微结构,血小板体外活化实验及非肥胖性糖尿病／严重联合免疫缺陷鼠异种体内移植实验评价扩增的巨核祖细胞功能。     

IL-6和IL-11对脐血CD34+细胞诱导分化为巨核细胞的影响

目的： 探讨白细胞介素－6(IL-6)和白细胞介素－11(IL-11)对脐血CD34+细胞诱导分化为巨核细胞及其产生血小板的影响。方法: 采用免疫磁珠法(MACS)分选8例健康产妇足月顺产的胎儿脐血中CD34+细胞，以含血小板生成素（TPO 50 μg/L）、白细胞介素-3（IL-3 10 μg/L）、干细胞因子（SCF 50 μg/L）的无血清培养基作为对照组，分别添加10 μg/L IL-6、IL-11、IL-6+IL-11作为实验组，培养14 d后观察结果。利用细胞计数仪检测单个核细胞数；流式细胞仪计数培养体系中的CD41+细胞和血小板；用倒置显微镜观察培养体系中的细胞生长情况；用显微镜和流式细胞仪观察凝血酶诱导后的血小板凝集情况。结果: 各实验组单个核细胞数与对照组无明显区别（P>0.05），而CD41+细胞和血小板数量明显多于对照组(P<0.05)。培养第14 d后倒置显微镜下可见实验组中血小板样颗粒物明显多于对照组，而且经凝血酶诱导后有明显血小板凝集。结论: IL-6和IL-11可诱导脐血中CD34+细胞分化为巨核细胞并产生功能性血小板。     

两阶段法体外诱导人动员外周血CD34~+细胞分化为巨核细胞

本研究提出两阶段法巨核细胞分化模型:首先将人外周血来源CD34+细胞在Cocktail或CC100两种增殖培养液中培养3、4、5或6 d,然后转入含有TPO和SCF的分化培养液中继续培养7、8或9 d。培养结束后通过比较细胞增殖、分化及成熟情况,优化培养条件。结果显示最优的诱导分化条件是,CD34+细胞在Cocktail培养液中扩增3 d后转入分化培养液中继续培养7 d;所获得的CD41+细胞和多倍体细胞数量是起始CD34+细胞的16倍和3倍;该条件下获得的CD41+和多倍体细胞数量显著高于常用的TPO法和TPO+SCF培养法。因此,运用本研究优化的两阶段培养模式能获得比单阶段直接诱导法更多的CD41+细胞和多倍体细胞,为巨核细胞和血小板相关的理论、临床研究提供新的更高效的巨核细胞体外扩增、分化模型。     

脐血CD34+CD19-造血干/祖细胞体外B细胞分化条件研究

目的:研究体外脐血造血干/祖细胞向B细胞分化的条件.方法:体外免疫磁珠分离纯化脐血CD34+CD19-造血干/祖细胞;在小鼠S-17基质细胞支持下,脐血CD34+CD19-造血干/祖细胞、T3、各种细胞因子共培养建立体外B细胞分化发育培养体系,诱导脐血CD34+CD19-造血干/祖细胞向B细胞分化;用流式细胞仪检测培养的B细胞.结果:T3、IL-7与小鼠S-17基质细胞共培养诱导CD34+CD19-造血干/祖细胞28天时,分化形成的B细胞数可达初始培养细胞的198倍,诱导细胞大部分表达CD10、CD19.结论:在选用的实验条件下,T3、IL-7与小鼠S-17基质细胞体外能诱导脐血造血干/祖细胞的B细胞分化.     

Daxx抑制K562细胞向巨核细胞分化

目的:观察过表达死亡结构域相关蛋白(Daxx)对K562细胞活力和向巨核细胞分化的影响。方法:建立稳定过表达Daxx的K562细胞,荧光显微镜观察、实时荧光定量PCR和Western blot检测Daxx的过表达效果,CCK-8法检测过表达后细胞活力的变化;佛波酯(PMA)诱导K562细胞向巨核细胞系分化,Western blot检测在K562细胞向巨核细胞分化过程中Daxx和p-ERK的表达变化,流式细胞术检测CD41和CD61的表达变化;PMA处理过表达Daxx的K562细胞,NBT还原实验检测细胞分化情况,流式细胞术检测过表达Daxx后CD41和CD61的表达变化,Western blot检测p-ERK的蛋白水平。结果:建立了稳定的过表达Daxx的K562细胞,过表达Daxx抑制K562细胞的活力。PMA诱导K562细胞向巨核细胞分化,CD41和CD61表达水平增高,同时p-ERK的蛋白水平升高,Daxx表达水平下降。过表达Daxx可以抑制K562细胞向巨核细胞分化,CD41和CD61表达降低,同时p-ERK的蛋白水平降低。结论:过表达Daxx可以抑制K562细胞生长及向巨核细胞分化,同时抑制ERK的磷酸化。     

γ干扰素上调人胎盘间充质干细胞PD-L1表达并增强其诱导脐血和外周血T细胞向IL-10~+T细胞的分化

目的比较人胎盘间充质干细胞(h PMSC)对脐血和外周血IL-10~+T细胞分化的调节作用,并探讨γ干扰素(IFN-γ)在其中的作用机制。方法应用酶消化法分离、培养h PMSC;反转录PCR和流式细胞术分别检测程序性死亡蛋白配体1(PD-L1)在h PMSC的表达;Ficoll密度梯度离心法分离脐血和健康成人外周血单个核细胞,并用红细胞花环法纯化T细胞;用植物血凝素(PHA)活化T细胞并与h PMSC进行共培养,流式细胞术分别检测在阻断型PD-L1 m Ab和IFN-γ预刺激的条件下h PMSC对脐血和外周血T细胞向CD4~+IL-10~+T细胞、CD8~+IL-10~+T细胞分化的诱导作用。结果 h PMSC可诱导脐血和外周血T细胞向CD4~+IL-10~+T细胞、CD8~+IL-10~+T细胞分化,且对外周血T细胞向IL-10~+T细胞分化的诱导能力明显高于对脐血T细胞向该细胞分化的诱导能力;IFN-γ预处理h PMSC后,h PMSC促进脐血和外周血T细胞向IL-10~+T细胞分化能力明显增强;h PMSC高表达PD-L1,IFN-γ可明显上调其在h PMSC上的表达;阻断PD-L1在h PMSC上表达后,脐血、外周血中CD4~+IL-10~+T细胞、CD8~+IL-10~+T细胞比例均明显降低。结论 h PMSC诱导外周血T细胞向IL-10~+T细胞分化的能力明显高于其诱导脐血T细胞向该细胞分化的能力。IFN-γ可通过上调PD-L1在h PMSC上的表达,增强h PMSC对脐血和外周血T细胞向IL-10~+T细胞分化的诱导能力。     

脐带间充质干细胞体外调节CD34+ CD126- 细胞向巨核系诱导的研究

目的:研究CD34~+CD126~-细胞向巨核细胞诱导分化及间充质干细胞对诱导过程的调节。方法:免疫磁珠分选获得CD34~+CD126~-细胞,设置3个实验组:无间充质组、间充质直接接触组和非直接接触组,以脐带血单个核细胞作对照,诱导向巨核细胞分化14 d,分别在7 d末与14 d末进行悬浮细胞数计数,流式测定CD34与CD41表型及镜下细胞形态观察。胶原蛋白凝胶培养基诱导CD34~+CD126~-细胞形成巨核细胞集落形成单位(CFU-Mk),12 d末CD41特异性抗原染色并镜下计数CFU-Mk。结果:(1)诱导7 d或14 d末,间充质组(CD34~+CD126~-细胞与MSC接触或非接触共培养组)获得悬浮细胞数均显著高于无间充质组(均为P0.001),但MSC接触与非接触组间获得悬浮细胞数差异无统计学意义(P0.05);(2)诱导7 d末,间充质组CD34阳性细胞数均显著大于无间充质组(均为P0.001),MSC接触组CD34阳性细胞数显著小于非接触组(P0.05),诱导14 d末,各组CD34阳性细胞率均下降至2%左右;(3)诱导7 d或14 d末,间充质组CD41阳性细胞数均显著大于无间充质组(均为P0.001),而MSC接触与非接触组间CD41阳性细胞数均无显著性差异(P0.05),CD34~+CD126~-细胞各组CD41阳性数均显著大于CBMCs的对应各组(均为P0.001);(4)CD34~+CD126~-细胞组诱导得到的大CFU-Mk(≥50个细胞)与中CFU-Mk(21～49个细胞)数量显著大于CBMCs组(P_(CFU-Mk;≥50 cells)0.01,P_(CFU-Mk;21-49 cells)0.05)。结论:脐带间充质干细胞的旁分泌作用对于巨核细胞分化过程中CD34~+细胞或CD41~+细胞的增殖均具有促进作用,但MSC的微环境能显著降低CD34~+细胞的增殖速率。诱导前筛选较为原始的巨核细胞祖细胞作为起始细胞对于巨核细胞诱导效率的提升具有显著作用。     

Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation

Inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs play an important regulatory role in immune cell activation. In addition, several studies suggest that these receptors are involved in the regulation of hematopoietic cell differentiation. Here, we have investigated the expression of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), an inhibitory receptor expressed on most peripheral blood leukocytes and on CD34+ hematopoietic progenitor cells, in neutrophil differentiation and activation. We found that although LAIR-1 was expressed on peripheral blood eosinophils, cell-surface expression on mature neutrophils was low, suggesting that LAIR-1 expression is regulated during granulocyte differentiation. Indeed, the promyeloid cell line HL-60 expressed LAIR-1, but the expression decreased during chemical-induced differentiation toward neutrophils. Similarly, in bone marrow-derived neutrophil precursors, the most immature cells expressed LAIR-1, and loss of LAIR-1 expression was associated with neutrophil maturation. LAIR-1 was re-expressed rapidly on the membrane of mature neutrophils upon stimulation with tumor necrosis factor alpha, granulocyte macrophage-colony stimulating factor, or N-formyl-methionyl-leucyl-phenylalanine, indicating that LAIR-1 may also regulate neutrophil effector function. Our studies suggest that LAIR-1 may play a regulatory role in differentiation and function of human granulocytes.     

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO-induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO-induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum-free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P-selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC-1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid-Schiff (PAS)-positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.     

Thrombopoietin combined with early-acting growth factors effectively expands human hematopoietic progenitor cells in vitro

Thrombopoietin (TPO) is established as a powerful stimulant of megakaryocyte differentiation and platelet production both in vivo and in vitro. In preparation for future transplantation of ex vivo expanded CD34+ hematopoietic progenitor cells (HPCs), we have examined the in vitro effect of TPO on cultures of HPC when combined with other early-acting hematopoietic growth factors (GFs) in an attempt to decrease post-transplant thrombocytopenia and accelerate engraftment. By adding TPO to all possible combinations of GM-CSF, IL-3, and c-kit ligand (CKL) in a suspension culture system, we found a significant increase in both relative and absolute numbers of cells in cultures containing TPO of the megakaryocytic lineage and CD34+ cells after 14 days of culture. The most efficient GF combinations for expansion of cell populations of the megakaryocytic lineage and HPCs were TPO, GM-CSF, and CKL, which increased the number of cells of the megakaryocytic lineage 78 fold and the number of CD34+ cells 1.8 fold. The number of CD34+ cells decreased in the cultures containing GM-CSF and CKL with no TPO present, and the number of cells of the megakaryocytic lineage was increased merely 27 fold. Based on our findings, we suggest adding cells from HPCs expanded in cultures containing TPO, GM-CSF, and CKL to unexpanded stem cells for stem cell transplantation.     

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.     

p40/LAIR-1 regulates the differentiation of peripheral blood precursors to dendritic cells induced by granulocyte-monocyte colony-stimulating factor

p40/LAIR-1, a member of the immunoglobulin superfamily, is a surface molecule broadly distributed among leukocytes which has been shown to down-regulate T and NK cell activation. In this study, we show that p40/LAIR-1 is highly expressed in CD14⁺ peripheral blood mononuclear cells (PBMC). When cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 – 14 days, CD14⁺ cells acquired morphologic and phenotypic features ( i.e. loss of CD14 and expression of CD80^bright and CD86^bright ) typical of dendritic cells (DC) and lost the expression of p40/LAIR-1. Engagement of p40/LAIR-1 (but not of CD58) by specific monoclonal antibodies prevented CD14⁺ PBMC differentiation into DC; when cultured in the presence of GM- CSF upon p40/LAIR-1 cross-linking, the resulting cells were CD14⁺ CD80^dull CD86^dull and displayed a macrophage-like morphology. We have recently demonstrated that peripheral blood CD14⁺ cells co-expressing the CD34 progenitor marker represent the circulating precursors of CD83⁺ DC. Herein we show that cross-linking of p40/LAIR-1 prevented the maturation of CD14⁺ CD34⁺ cells into CD83⁺ DC. This effect appears to be consequent to the impairment of GM-CSF receptor-mediated activation signaling. Indeed, triggering of GM-CSF receptors in both CD14⁺ and CD14⁺ CD34⁺ cells led to increases in the intracellular free calcium concentrations which were inhibited by p40/LAIR-1 engagement. Taken together, these data suggest a possible regulating role played by p40/LAIR-1 in the process of differentiation from peripheral blood precursors into DC induced by GM-CSF.     

Regulated expression of the inhibitory receptor LAIR-1 on human peripheral T cells during T cell activation and differentiation

The leukocyte-associated Ig-like receptor-1 (LAIR-1) is capable of inhibiting immune cell function through interaction with collagens. LAIR is expressed on the majority of peripheral blood mononuclear cells. The abundant expression of both receptor and ligand calls for regulatory mechanisms to relieve the continuous interaction between collagens and LAIR-1. This regulation may occur at the expression level of the receptor. Here, we report that LAIR-1 is indeed differentially expressed during human T cell differentiation. Naive CD4(+) and CD8(+) T cells as well as CD8(+) T cells of the effector phenotype express higher levels of LAIR-1 compared to memory T cells. In vitro stimulation revealed a decrease in LAIR-1 expression upon activation, and the lower LAIR-1 expression on CD127(-) T cells suggests that activation-induced down-modulation of LAIR-1 may also occur in vivo. Furthermore, crosslinking of LAIR-1 on primary T cells results in an inhibition of T cell function. Our data suggest that regulated expression of LAIR-1 and the subsequent change in the threshold for activation may be a mechanism to modulate inhibition of the immune system.