The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L.

This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.     

Delta-1对IL-6R+髓系祖细胞分化的影响

目的 探讨Notch配体Delta-1在髓系造血细胞分化过程中对膜结合和可溶性IL-6受体所介导信号的调节作用.方法 分离正常脐血单核细胞,然后利用CD34免疫磁珠试剂盒和FACSVantage流式细胞仪从所获单核细胞中拣选CD34 CD38-细胞;将CD34 CD38-细胞利用SCF、Flt3L、TPO和IL-3(4GFs)培养7 d,用CD36免疫磁珠试剂盒分离掉培养细胞中的CD36 红系祖细胞,然后用FACSVantage流式细胞仪将细胞再次拣选出CDl15- CD14- CD1a- IL-6R 表型的细胞,将这种表型的细胞用含有4GFs、4GFs IL-6或4GFs FP6培养基在有或无Delta-1存在的条件下进行培养11 d,并对CD15 粒细胞、CD14 单核细胞和CD14-CD1a 树突状细胞进行计数.结果 发现所有CD15、CD14或CD1a阳性的细胞均表达IL-6R;IL-6和FP6可促进CD15 细胞的分化;Delta-1在无IL-6和FP6存在时对CD15 细胞的分化表现出轻度的抑制作用;在IL-6和FP6存在时对CD15 粒细胞的分化表现出明显的抑制作用;相反,IL-6和FP6抑制CD14-CD1a 细胞的分化,而Delta-1促进CDl4-CD1a 细胞的分化.结论 Delta-1可通过抑制mIL-6R-和sIL-6R所介导的IL-6生物学效应抑制IL-6R 髓系祖细胞分化为粒细胞和单核细胞,但促进其分化为树突状细胞.     

HGF activates signal transduction from EPO receptor on human cord blood CD34+/CD45+ cells

Hepatocyte growth factor (HGF) is a multifunctional cytokine with early hematopoiesis-stimulatory activity. Here, we focus on its erythropoiesis-stimulatory effect on highly purified human hematopoietic progenitor cells (CD34+/CD45+ cells) derived from the cord blood. In immunoblot analyses, c-met protein (a receptor of HGF) was detected in the CD34+/CD45+ cells, although the expression levels were different among samples. The c-met expression was facilitated by incubation of the cells with stem cell factor (SCF) or interleukin 3 (IL-3), even if the expression level had been low. IL-6, G-CSF, or erythropoietin (EPO) did not show such a stimulatory effect on the c-met expression of the cells. When HGF was added to the CD34+/CD45+ cells in the presence of SCF, the numbers of CD36+/CD11b- cells (very early erythroid lineage cells) and BFU-E increased. EPO-dependent tyrosine phosphorylation of Stat 5 also increased, but the EPO receptor (EPO-R) expression remained unchanged in the CD34+/CD45+ cells treated with SCF + HGF. Our present study suggests that stimulation of the HGF/c-met signal is concomitant with induction of c-met protein by SCF. The subsequent enhancement of signal transduction via the activation of Stat 5 from the EPO-R plays a crucial role in the commitment of hematopoietic stem cells into erythroid lineage cells.     

Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia

We examined the effects of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), and thrombopoietin (TPO), alone or in combination, on the generation of neutrophils by bone marrow (BM) cells from three patients with severe congenital neutropenia (SCN) through the use of a serum-deprived liquid culture system. Synergistic effects of G-CSF and SCF on the neutrophil production by BM CD34+CD38+c-kit+ cells were observed in SCN patients as well as in normal controls. The addition of TPO to the culture containing G-CSF and SCF further augmented the growth of neutrophils in the two groups. Single-cell culture experiments revealed that the three-factor combination caused increases in both the number and size of neutrophil colonies compared with G-CSF + SCF in normal BM cells, whereas only a significant increment in the colony size was observed in SCN patients. Even in the presence of SCF or SCF + TPO, the concentrations of G-CSF necessary for the substantial production of neutrophils by CD34+CD38+c-kit+ cells were higher in two patients compared with the levels obtained by normal control cells. In addition, TPO did not accelerate the maturation of neutrophilic cells supported by G-CSF + SCF. When BM CD34+CD38-c-kit+ cells were targeted, the addition of TPO to the culture containing G-CSF and SCF was required for significant neutrophil colony growth in the two groups. These results suggest that TPO enhances the G-CSF-dependent neutrophil production with the aid of SCF in this disorder.     

Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.     

Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of notch signaling-related genes in CD34+ cells

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1- and Jagged1-expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34(+) CD38(+) cells. Jagged1 increases the number of bipotent colony-forming unit-granulocyte macrophage (CFU-GM) and unipotent progenitors (CFU-granulocytes and CFU-macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU-GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34(+) cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1-CD34(+) cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling-related genes in CD34(+) CD38(+) cells as they develop in Jagged1- or Delta1-stromal cell environments, which appear to reflect sequential maturational stages of CD34(+) cells into distinct cell lineages.     

Selective generation of different dendritic cell precursors from CD34+ cells by interleukin-6 and interleukin-3

There is a growing interest in generating dendritic cells (DCs) for using as vaccines. Several cytokines, especially stem cell factor (SCF) and FLT3-ligand (FL), have been identified as essential to produce large numbers of myeloid precursors and even to increase DC yield obtained by the action of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). However, there are few studies on the effect of the early-acting cytokines, commonly used to expand CD34+ progenitor cells, on DC generation. We report here that in the absence of serum, SCF, FL, and thrombopoietin (TPO) plus interleukin-6 (IL-6) and SCF, FL, and TPO plus IL-3 were able to generate CD14+CD1a- and CD14- CD1a+ myeloid DC precursors from CD34+ cells, but IL-6 had an inhibitory effect on the generation of CD14- CD1a+ cells. Both DC precursors differentiated into mature DCs by GM-CSF, IL-4, and TNF-alpha, and DCs obtained from both types of culture exhibited equal allostimulatory capacity. CD1a+ DCs generated could be identified on the basis of DC-specific intracellular adhesion molecule-grabbing nonintegrin (DC-SIGN) expression, a novel C-type lectin receptor expressed on dermal DCs but not on Langerhans cells. In addition, the inclusion of IL-3 to the culture medium induced the appearance of CD13- cells that differentiated into plasmacytoid DC (DC2) on the addition of TNF-alpha, allowing the identification of developmental stages of DC2. Like true plasmacytoid DCs, these cells secreted interferon-alpha after TLR9-specific stimulation with a specific CpG nucleotide.     

Interleukin-3 does not affect the differentiation of mast cells derived from human bone marrow progenitors

Although IL-3 is commonly used for culture of human progenitor-derived mast cells together with Stem cell factor (SCF) and IL-6, the effect of IL-3 on human mast cell differentiation has not been well elucidated. Human bone marrow CD34+ progenitors were cultured for up to 12 weeks in the presence of rhSCF and rhIL-6 either with rhIL-3 (IL-3 (+)) or without rhIL-3 (IL-3 (-)) for the initial 1-week of culture. Total cell number increased at 2 weeks in IL-3 (+), as compared to IL-3 (-), but changes in the appearance of mast cells were delayed. When IL-3 was present for the initial 1-week culture, granules looked more mature with IL-3 than without IL-3. However, tryptase and chymase contents, and surface antigen expression (CD18, CD51, CD54, and CD117) were not altered by IL-3. Surface expression and mRNA level of FcepsilonRIalpha and histamine release by crosslinking of FcepsilonRIalpha did not differ from one preparation to the next. GeneChip analysis revealed that no significant differences were observed between IL-3 (+) and IL-3 (-) cells either when inactivated or activated by aggregation of FcepsilonRIalpha. These findings indicate that initial incubation of human bone marrow CD34+ progenitors with IL-3 does not affect the differentiation of mast cells.     

Megakaryocytopoiesis in vitro: from the stem cells' perspective

Megakaryocytopoiesis is a complex network regulated by different megakaryocyte (MK)-stimulating factors (i.e., thrombopoietin [TPO], stem cell factor [SCF], interleukin 3 [IL-3], IL-6, IL-11 and GM-CSF). Although all of these factors can affect human and murine megakaryocytopoiesis at different levels of MK development, the effect on very primitive hematopoietic stem cells (HSC) is not well understood. We have further characterized the in vitro biological activity of recombinant murine TPO, SCF and IL-3 on the maturation and proliferation of MK progenitors from different murine primitive hematopoietic cells in a fibrin clot system under serum-free conditions. Neither TPO nor SCF alone induced MK colony formation (CFU-MK) from Lin- Sca+ cells. However, isolated large and mature MKs were observed in the presence of TPO. In contrast, IL-3 exerted a potent effect on CFU-MK formation from Lin- Sca+ cells. On this population of HSC, a significant increase of large MK colonies with mature MK were obtained under those conditions in which TPO was combined with IL-3 or SCF plus IL-3. Similar results were obtained with murine bone marrow cells enriched by primitive progenitors from day 3 post-5-fluorouracil treated mice (5-FUBMC). In contrast, TPO-sensitive precursors were detected in fetal liver cells (FLC). These cells differentiate and proliferate to MK progenitors in the presence of TPO. A significant increase in the number of CFU-MK was induced when TPO was combined with either IL-3 or SCF. On these populations of primitive hematopoietic progenitors, IL-3 induced both the proliferation and differentiation of MK progenitors. Because erythropoietin and TPO share similarities between their molecules and their receptors, we studied whether these growth factors may modulate megakaryocytopoiesis from FLC. Flow cytometry analysis of FLC expressing erythroid markers demonstrated that these cells expressed c-Mpl receptor. In our in vitro studies, although EPO by itself did not induce MK colonies from FLC, it enhanced the proliferative activity of TPO. High ploidy and proplatelet-shedding MK were observed in Lin- Sca+ cells, 5-FUBMC and FLC stimulated with TPO alone or in combination with other MK-stimulating factors. Based on these observations, we propose that TPO, IL-3 and SCF constitute early MK-acting factors with differential proliferative and differentiative activities on murine stem cells. TPO by itself does not appear to be involved in the proliferation of MK progenitors from bone marrow HSC. TPO appears to induce in these cells the commitment toward MK differentiation. However, this growth factor may enhance the proliferative activity of IL-3. IL-3 is an early MK-stimulating factor able to induce in vitro the proliferation and differentiation of MK progenitors from HSC.     

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.     

Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels

Ex vivo expansion of human umbilical cord blood cells (HUCBC) is explored by several investigators to enhance the repopulating potential of HUCBC. We performed experiments using either Ficoll-separated or CD34+-selected HUCBC from the same donation in serum-free medium. CD34-purified HUCBC were cultured on either human umbilical vein endothelial cells (HUVEC) or irradiated bone marrow-derived stroma cells (BMSC) with addition of different cytokines. In addition, we tested the expansion of HUCBC in culture vessels with continuous rotation. CD34 enrichment led to a significant increase in the expansion factor of CD34+ cells compared with unmanipulated HUCBC. BMSC were more efficient in amplifying early progenitors than HUVEC. Optimum results were reached by a combination of SCF, FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml. No significant improvement in the expansion of CD34+/38- primitive progenitors could be obtained with other combinations. Addition of megakaryocyte-derived growth and development factor to each growth factor cocktail improved the expansion results. Continuous rotation of culture vessels did not ameliorate the expansion rate of the analyzed subsets. Culture conditions separating stroma and HUCBC by a semipermeable membrane improved the expansion factors of CD34+, CD34+/38-, and CD34+/41+ cells and CFU-GM compared with contact cultures. These data might be useful when designing culture systems for clinical scale ex vivo expansion of HUCBC.     

Notch信号介导共培养脐带间充质干细胞和造血干细胞的相互作用

目的:探讨脐带间充质干细胞(UC-MSC)体外与造血干细胞共培养后Notch信号分子的改变。方法:通过胶原酶消化方法分离UC-MSC,通过流式细胞仪检测以及成脂、成骨和成软骨诱导鉴定UC-MSC具备间充质干细胞的特性。进而,将UC-MSC与脐血CD34+造血干细胞(HSC)体外培养,实时PCR方法检测MSC及CD34+细胞表面Notch配体及受体表达以及表达是否存在变化;在共培养体系中加入Notch信号阻滞剂DAPT(γ-secretase抑制剂),比较Hes-1基因活化状态的改变。结果:体外实验显示:UC-MSC在形态学、细胞表面表型和诱导分化能力上均具备间充质干细胞的特性。UC-MSC及CD34+细胞表面存在Notch信号配体及受体的表达,共培养后Jagged 1、Notch1基因表达明显增加;共培养后CD34+细胞中的Hes-1基因表达明显增加而加入DAPT后Hes-1基因表达未检出明显改变。结论:UC-MSC支持造血中,Notch信号可能发挥重要作用。     

Interleukin-15 Promotes the Commitment of Cord Blood CD34^＋ Stem Cells into NK Cells

Naturalkiller(NK)cellsweresubgroupoflymphocytes thatplayedanessentialroleinthecellularbasedimmune defenseagainstvirus infectedandtransfectedcells.His torically,inteleukin 2(IL 2)wasregardedasthenatural activatorforNKcells.Recently,itwasdiscoveredthat IL …     

重组人IL-12和SCF促进新生儿脐带血中CD34~+细胞的分裂增殖

目的分析IL-12及SCF对CD34+细胞分裂增殖及IL-12Rβ的表达。方法分离正常足月产新生儿脐带血与健康成年人外周血中的单个核细胞,采用流式细胞术(FACS)比较2组中CD34+细胞的频率,分析IL-12及SCF对CD34+细胞的作用。结果健康成年人外周血中CD34+细胞平均频率为0.13%,显著低于新生儿脐带血中CD34+细胞的平均频率1.53%(P<0.001)。进一步研究表明,SCF可以促进CD34+细胞表达CD25;IL-12及SCF可以显著促进CD34+细胞的分裂增殖以及IL-12Rβ1的表达。结论新生儿脐带血中CD34+细胞的频率明显高于成年人外周血中CD34+细胞的频率。IL-12及SCF可以促进CD34+细胞的分裂增殖以及IL-12Rβ1的表达。     

天花粉蛋白及其肽段诱导的免疫抑制依赖APC表面Notch配体的表达

新近发现,Notch信号途径参与调节外周成熟T细胞及其亚群的分化和功能发挥。本研究应用天花粉蛋白及其衍生肽处理骨髓来源的小鼠树突状细胞(DC),检测Notch配体家族分子的表达及DC对CD8+T细胞分泌细胞因子的影响。结果表明,天花粉蛋白或其衍生肽PB处理DC可使Notch配体Jagged1、Delta1分子表达明显增加,并改变CD8+T细胞细胞因子分泌格局,明显抑制Th1相关细胞因子IFN-γ的分泌,而Th2相关细胞因子IL-4和IL-10分泌明显增加。Notch信号的阻断剂可以部分逆转Tk及肽段的抑制作用。表明天花粉蛋白及其衍生肽可诱导一群具有抑制能力的CD8+T细胞,该作用依赖于DC表面Notch配体的表达。     

FK228促进IL-3介导的人红系前体细胞的增殖与分化

目的:探讨组蛋白脱乙酰化酶抑制剂FK228在IL-3介导的人红系前体细胞增殖与分化中的调节作用.方法:经粒细胞集落刺激因子动员的肿瘤患者外周血单核细胞中分离CD34 细胞,用含干细胞生长因子(SCF)、白细胞介素3(IL-3)或SCF IL-3及不同浓度FK228的无血清培养液培养7 d,分别用抗CD14、GPA、CD15及CD36单克隆抗体(mAb)染色并行流式细胞术(FCM)检测;将CD34 细胞用含SCF、IL-3或SCF IL-3及FK228的半固体培养液培养,并行细胞集落分析;将CD34 细胞用含SCF IL-3的培养液培养7 d,然后分离CD36 GPA-细胞,将细胞用含有IL-3 FK228的半固体培养液中培养,并行细胞集落分析.结果:FK228以一种剂量依赖方式促进CD36 细胞的产生,对CD14 细胞及CD15 细胞的产生无明显影响;0.5 μg/L FK228可明显增加含IL-3培养体系中CD36 细胞的数量;FK228可明显增加含IL-3的半固体培养基中集落形成的数量及组成单个集落的细胞数;FK228可明显促进CD36 GPA-细胞在含IL-3的半固体培养基中形成红细胞集落.结论:FK228可促进白细胞介素3介导的人红系前体细胞的增殖与分化.     

The role of Notch receptor expression in bile duct development and disease

Mutations in the Jagged1 gene, a ligand for the Notch signalling pathway, have been implicated in the pathogenesis of Alagille syndrome (AGS), resulting in bile duct paucity. Recently, a mouse model for AGS suggested that abnormalities of the Notch2 receptor, as well as of Jagged1, may be present. Expression patterns of Notch receptors have not been described in the developing human liver or in paediatric liver. The expression of Notch receptors and ligands was examined in fetal, paediatric normal, and diseased human liver by RT-PCR and immunohistochemistry. RT-PCR showed Notch1-4 mRNA expression to be present. In fetal liver, Notch3 protein was expressed on mesenchymal cells, closely adjacent to ductal plate cells that expressed Jagged1. In paediatric normal liver, Notch1 and Notch2 were present on mature bile duct cells. Notch expression was altered in disease, with distinct differences in AGS from extrahepatic biliary atresia (EHBA) and alpha1-anti-trypsin deficiency (alpha1AT). In AGS, where extensive ductular reaction was present, Jagged1 was expressed on ductular reactive cells (DRCs), along with marked Notch2 and Notch3 staining. Where there was ductular paucity, Notch2 and Notch3 were not expressed on remaining biliary epithelial cells. In EHBA and alpha1AT, Notch receptor expression was not seen on DRCs. Instead, Notch2 and Notch3 were expressed by stromal cells. In all diseases, Notch3 was expressed on neovessels in portal tracts and cirrhotic fibrous septa. In conclusion, Notch3 is expressed in close proximity to Jagged1 at the time of ductal plate formation, suggesting that Notch3 is important for bile duct development. The expression of both Notch2 and Notch3 in AGS on DRCs confirms that these receptors may be important in the pathogenesis of this disease. Further studies are required to investigate the presence of Notch2 and Notch3 at other periods in liver development and to clarify the role of Notch signalling in paediatric cholestases.     

细胞因子短期扩增对造血干/祖细胞黏附和迁移能力的影响

目的：探讨干细胞因子（SCF）+白细胞介素-6（IL-6）短期扩增对CD34+造血干/祖细胞黏附和迁移能力的影响。方法:用密度剃度离心的方法分离脐血CD34+细胞，经SCF和IL-6孵育48 h，用CCK-8方法检测CD34+细胞增殖能力；用流式细胞仪检测处理前后的CD49d（VLA-4）、CD11a（LFA-1）、CD62L（L-selectin）及CD184（CXCR4）的表达。用纤连蛋白（FN）包被96孔板，检测经或未经因子扩增的CD34+细胞的黏附能力。扩增的CD34+细胞悬浮于transwell培养板的上层，下层添加基质细胞衍生因子（SDF-1），流式细胞仪检测迁移细胞数，计算迁移率。结果:经SCF+IL-6处理48h后CD34+细胞扩增近3倍；表达CD49d、CD11a、CD62L及CD184的CD34+细胞的百分数分别由原来的26.34%±5.37%、17.63%±4.57%、46.38%±6.61%和9.58%±1.56%增加到65.67%±8.72%、56.67%±6.34%、84.76%±9.57%和19.32%±3.64%（P<0.01）。扩增后的CD34+细胞对FN的黏附能力及在SDF-1诱导下的迁移作用都显著增强（P<0.01）。结论:SCF+IL-6短期扩增CD34+ 造血干/祖细胞显著增加细胞的黏附能力，增加SDF-1诱导的迁移作用，可能是SCF+IL-6促进归巢的主要机制之一。     

重组人IL-15促人脐血CD34+造血干细胞定向分化为NK细胞

目的 探讨IL－15促CD34^ 造血干细胞定向分化为NK细胞的作用。方法 采用MACS法分离脐血中CD34^ 细胞，分别用IL－15、SCF（造血干细胞因子）和IL－15 SCF体外培养，流式细胞仪测定CD3、CD16、CD56分子，MTT法检测NK细胞杀伤活性。结果 IL－15可以促进CD34^ 细胞分化为以CD56^ CD16^-为主的NK细胞，SCF具有较强的协同作用。结论 IL－15具有促使NK细胞定向分化的作用。     

Differential regulation of Notch ligands in dendritic cells upon interaction with T helper cells

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta-like and Jagged, in antigen-presenting cells. However, whether stimuli other than pathogen-derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta-like 4 and Jagged 2 expression in bone marrow-derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta-like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL-4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2-promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL-4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL-4. These data suggest that Notch ligands are differentially regulated in DCs: Delta-like 4 is regulated by T helper cells and by pathogen-derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen-derived Th2-promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs.