人类脐血与正常骨髓CFU—F特性的比较研究

用液体培养法对１４份人类脐血和１０份正常骨髓ＣＦＵ－Ｆ的生长特必，形态学，细胞化学及超微结构等进行了研究，结果除生长特性及集落数不同外，两者无明显差异。说明脐血含有基质细胞，在脐血ＣＦＵ－Ｆ培养中，还发现一种来源，性质不明的细胞，提示脐血具有不同于骨髓之处。     

脐血输注对急性白血病患者骨髓CFU－GM体外培养及血浆CSA、BPA的影响

以自身对比方法观察急性白血病化疗后骨髓衰竭患者输注脐血和外周血后，骨髓粒、单细胞系集落形成单位（ＣＦＵ－ＧＭ）、血浆集落刺激活性（ＣＳＡ）、爆式集落刺激活性（ＢＰＡ）及外周血白细胞计数（ＷＢＣ）的变化。结果脐血输注后可使患者ＣＦＵ－ＧＭ体外培养显著提高，ＣＳＡ、ＢＰＡ水平显著下降并趋于正常，可在输血量减少情况下，使ＷＢＣ显著增高。     

脐血造血细胞的体外扩增及分化特征

目的：了解造血生长因子的不同组合对人脐血ＣＤ３４＋细胞的扩增效应及分化特性。方法：采用人脐血单个核细胞（ＭＮＣ）经ｒｈＩＬ－１,ｒｈＩＬ－３,ｒｈＩＬ－６,ｒｈＧ－ＣＳＦ,ｒｈＧＭ－ＣＳＦ和ｒｈＳＣＦ的不同组合进行体外扩增培养,动态观察了不同细胞因子组合对有核细胞总数的扩增数量,应用流式细胞技术（ＦＡＣＳ）动态分析了造血细胞的表面标志,并对液态扩增后造血细胞的粒－巨噬集落形成率（ＣＦＵ－ＧＭ）进行了动态观察。结果：经上述６种细胞因子培养２０ｄ,有核细胞总数增殖４４倍,液态扩增培养８ｄ后,ＣＦＵ－ＧＭ比原代ＭＮＣ增加１４．７４倍,扩增１８ｄ后,ＣＦＵ－ＧＭ明显减少,仅为原代ＭＮＣ的６．４２倍,且集落小于前者。扩增培养至６～８ｄ时,ＣＤ３４＋细胞总数是原代 ＭＮＣ的１２７．７９～１９６．４０倍,１８ｄ时下降至１０１．５１倍。结论：外源性造血生长因子可使脐血ＣＤ３４＋细胞及ＣＦＵ－ＧＭ产生明显的扩增效应。     

脐带血清在培养人CFU-GM中作用探讨

脐带血清在培养人ＣＦＵ－ＧＭ中作用探讨王兰琴，曲垣瑞，胡治黄，裴福生在脐血造血细胞特性和造血潜能研究中，发现脐血中富含造血干／祖细胞，及多种造血刺激因子［１］。我们在体外培养正常人ＣＦＵ－ＧＭ时，加入脐带血清，试图了解其中的造血调控规律，现将实验结果...     

人骨髓,脐血CD34^＋干细胞体外扩增的树突状细胞的表型及 …

目的：从人骨髓造血前体细胞体外培养扩增树突状细胞（ＤＣｓ）,测定其表型及Ｔ细胞刺激活性。方法：采用Ｍｉｎｉ－ＭＡＣＳ分离技术,从正常人骨髓、脐血分离ＣＤ３４＾＋造血干细胞,体外以重组ｈＧＭ－ＣＳＦ,ｈＴＮＦ－α,ｈＩＬ－３诱导培养２周,流式细胞术检测扩增细胞的表面表型及细胞内ＩＬ－１２的表达,体外同种混合淋巴细胞反应检测扩增ＤＣｓ的Ｔ细胞刺激活性。结果：从正常人骨髓、脐血分离得到高纯度（〉９０％）     

骨髓增生异常综合征患者体外CFU-GM、CFU-L培养和细胞遗传学研究

研究了４２例骨髓增生异常综合征患者体外ＣＦＵ－ＧＭ、ＣＦＵ－Ｌ培养和细胞遗传学改变。结果显示，患者ＣＦＵ－ＧＭ明显低于正常对照组（Ｐ＜０．０１），但高于再生障碍性贫血（ＡＡ）（Ｐ＜０．０１），ＣＦＵ－Ｌ高于正常对照组和ＡＡ组（Ｐ＜０．０１）。患者５２．４％存在染色体异常，常见为：＋８、－２２、－Ｘ、－Ｙ、－２０、－７／７ｑ－。ＣＦＵ－ＧＭ减低、ＣＦＵ－Ｌ增高和染色体异常者疗效较正常者差。表明ＣＦＵ－ＧＭ、ＣＦＵ－Ｌ体外培养和细胞遗传学检查可作为ＭＤＳ协助诊断和预测疗效的指标之一。     

脐血输注对急性白血病患者骨髓CFU—GM体外培养及血浆CSA,BPA的影响

以自身对比方法观察急性白血病化疗后骨髓衰竭患者输注脐血和外周血后，骨髓粒、单细胞系集落形成单位（ＣＦＵ－ＧＭ）、血浆集落刺激活性（ＣＳＡ）、爆式集落刺激活性（ＢＰＡ）有外周血白细胞计数（ＷＢＣ）的变化。结果脐血输注后可使患者ＣＦＵ－ＧＭ体外增减显著提高，ＣＳＡ、ＢＰＡ水平显著下降并趋于正常，可在输血量减少情况下，使ＷＢＣ显著增高。     

骨髓增生异常综合征患者体外CFU—GM,CFU—L培养和细胞遗传学研究

研究了４２例骨髓增生异常综合征患者体外ＣＦＵ－ＧＭ、ＣＦＵ－Ｌ培养和细胞遗传学改变。结果显示，患者ＣＦＵ－ＧＭ明显低于正常对照组（Ｐ〈０．０１），但高于再生障碍性贫血（ＡＡ）（Ｐ〈０．０１），ＣＦＵ－Ｌ高于正常对照组和ＡＡ组（Ｐ〈０．０１）。患者５２．４％存在染色体异常，常见为：＋８、－２２、－Ｘ、－Ｙ、－２０、－７／７ｑ＾－。ＣＦＵ－ＧＭ减低、ＣＦＵ－Ｌ增高和染色体异常者疗效较正常者差。表明ＣＦ     

肿瘤坏死因子,干扰素和免疫活性肽对癌瘤患者骨髓粒—巨造血祖细…

应用ＣＦＵ－ＧＭ软琼脂培养技术，在体外观察了不同浓度的ｒｈ－ＴＮＦ，ｒｈ－ＩＦＮα－２和ＩＡＰ对９例恶性淋巴瘤和１例睾丸癌患者的骨髓粒、巨造血祖细胞生长的影响。结果表明，在培养体系中，加入ｒｈ－ＴＮＦ５０和１００ｕ／ｍｌ，其ＣＦＵ－ＧＭ较空白对照均明显增多（Ｐ〈０．０５）。ｒｈ－ＩＦＮα－２１０００ｕ／ｍｌ可使ＣＦＵ－ＧＭ减少（Ｐ〈０．０５）。ＩＡＰ似乎对ＣＦＵ－ＧＭ无影响（Ｐ〉０．０５）。集落形     

肿瘤坏死因子、干扰素和免疫活性肽对癌瘤患者骨髓粒－巨造血祖细胞的影响

应用ＣＦＵ－ＧＭ软琼脂培养技术，在体外观察了不同浓度的ｒｈ－ＴＮＦ，ｒｈ－ＩＦＮα－２和ＩＡＰ对９例恶性淋巴瘤和１例睾丸癌患者的骨髓粒－巨造血祖细胞生长的影响。结果表明，在培养体系中，加入ｒｈ－ＴＮＦ５０和１００ｕ／ｍｌ，其ＣＦＵ－ＧＭ较空白对照均明显增多（Ｐ＜０．０５）。ｒｈ－ＩＦＮα－２１０００ｕ／ｍｌ可使ＣＦＵ－ＧＭ减少（Ｐ＜０．０５）。ＩＡＰ似乎对ＣＦＵ－ＧＭ无影响（Ｐ＞０．０５）。集落形态学显示，ｒｈ－ＴＮＦ和ＩＡＰ均能增加巨噬细胞为主形成的集落（Ｐ＜０．０１）。我们认为，该实验为骨髓移植及肿瘤患者在选择应用这些细胞因子上提供了一定的佐证。     

Differentiation and maturation of growth factor expanded human hematopoietic progenitors assessed by multidimensional flow cytometry.

Non-adherent cord blood and bone marrow mononuclear cells were analyzed by multiparameter flow cytometry before and at day 2, 4, 7, and 11 of culture in recombinant interleukin 3 (IL-3) and granulocyte colony-stimulating factor (G-CSF, cord blood) or stem cell factor (SCF), IL3 and granulocyte-macrophage colony-stimulating factor (GM-CSF, BM) to assess the differentiation and maturational pathway of myeloid cells. Before cell culture cord blood contained progenitor cells (CD34+) in various differentiation stages (CD38(-)----CD38bright), mature lymphocytes, monocytes, and neutrophils, but no immature neutrophils and immature monocytes. During cell culture, all CD34+ cells acquired the CD38 antigen between day 2 and 5 of cell culture, the CD34 antigen was lost between day 5 and 11 of cell culture. Differentiation of cells into the myeloid cell lineage was characterized by the acquisition of both CD33 and CD71. The latter is indicative for the active proliferation of these cells. Maturation of the cells into the neutrophilic pathway was indicated by the acquisition of first the CD15 antigen followed by CD11b and CD16 respectively. Whereas maturation of the cells into the monocytic pathway was indicated by the acquisition of first CD11b followed by CD14 and a dim expression of both CD15 and CD16. In normal bone marrow, cells of various maturational stages are already present before cell culture. During cell culture differentiation of cells into the myeloid lineage and maturation of the cells along the monocyte and neutrophilic lineage followed identical pathways as was observed before cell culture. Differentiation and maturational pathways of cord blood and adult bone marrow were identical. The results confirm the surface-antigen-defined pathways of myeloid cell differentiation described previously for non-cultured normal bone marrow aspirates. The detailed assessment of cell maturation and differentiation of cultured cells by multidimensional flow cytometry permits the determination of the specific effects of various recombinant human growth factors on myeloid cells.     

非血缘脐血造血干细胞移植的现状

 脐血是骨髓和外周血干细胞以外的新的干细胞主要来源，脐血移植特别是非血缘脐血移植（UCBT）的数量逐年增加。UCBT存在的主要问题在于脐血的造血干／祖细胞（HSC／HPC）数量少，可能导致植入延迟或植入失败。临床上采用多种方法来提高脐血HSC／HPC的数量，包括双份脐血输注、减低强度的预处理、体内体外扩增脐血细胞、同时输注单倍体血缘供者CD+34细胞或间充质干细胞（MSC）、或将脐血细胞直接注入骨髓等，取得了良好的疗效。UCBT将会成为更多患者的治疗选择。     

Bone and bone marrow interactions: hematological activity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide. II. Action on human hematopoietic stem cells

Osteogenic growth peptide (OGP) is a peptide exerting regulatory effects on the bone and on bone marrow. The carboxy-terminal pentapeptide (OGP10-14) is the biologically active portion of OGP. We evaluated OGP10-14 hematopoietic activity performing colony-forming tests on human stem cells derived by bone marrow, peripheral blood and cord blood. Granulocyte-macrophage colony-forming unit (CFU) were significantly increased in OGP10-14-treated samples, while granulocyte-erythrocyte-monocyte-megakaryocyte CFU and burst-forming unit (BFU) erythroid were increased only in the cord blood test.Moreover, OGP10-14 preserves stem cells self renewal potential in long-term culture (LTC) initiating cells and acts directly on CD34+ enriched cells or by increasing activity of stem cell factor (SCF) and granulocyte-megakaryocyte colony-stimulating factor.     

重组人集落刺激因子对脐带血和骨髓造血祖细胞体外培养的影响

分别应用单层琼脂培养和甲基纤维素培养方法，同期观察了重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）、重组人粒单细胞集落刺激因子（ｒｈＧ－ＣＳＦ）、重组人白细胞介素－３（ｒｈＩＬ－３）和红细胞生成素（ＥＰＯ），对脐带血和骨髓造血祖细胞的影响。结果显示：脐带血中含有丰富的造血祖细胞，其早期的造血祖细胞数量与骨髓相似，脐带血的造血祖细胞的增殖能力更强。     

rhIL-17对小鼠造血前体细胞和人脐血CD34+干细胞分化发育的影响 *

目的:探讨重组人白细胞介素-17(Interleukin-17,IL-17)对小鼠骨髓造血前体细胞和人脐血来源的CD34~ 干细胞生长发育的影响.方法:采用常规方法采集小鼠造血前体细胞;采用Mini-MACS分离技术,从正常人脐血分离人CD34~ 干细胞.体外加入IL-17和/或GM-CSF、IL-4培养分离的前体细胞,应用流式细胞仪检测其表型,采用ELISA法检测了其分泌的IL-12水平,通过[~3H]-TdR掺入法测定其刺激同种异体T淋巴细胞增殖的能力.结果:IL-17促进了小鼠骨髓来源的未成熟DC表达Ia,B7-2等免疫分子,促使其分泌较高水平的IL-12,该细胞也能刺激同种异体T细胞有效增殖,表现出了成熟DC的特征.IL-17单独培养9d促使人脐血CD34~ 干细胞扩增了2倍,部分细胞高表达CD1a及B7-2,低表达HLA-DR,未检测到CD83的表达.该细胞能促使同种异体T细胞增殖,但作用较弱;而rhIL-17与GM-CSF联合培养后扩增了14倍,培养细胞中CD1a、B7-2阳性细胞的比例明显升高,且此细胞刺激同种异体T细胞增殖的能力较强.结论:IL-17体外可促进小鼠骨髓造血前体细胞来源的DC成熟;与GM-CSF联合培养既能促进CD34~ 干细胞增殖,又能使之获得DC特征,初步提示IL-17与GM-CSF联合作用可促进CD34~ 干细胞向DC分化.     

Fresh peripheral blood mononuclear cell preparations are a better starting material than bone marrow after cryopreservation for immunomagnetic harvesting of CD34(+) hematopoietic cells

Immunomagnetic separation using anti-CD34 monoclonal antibodies and paramagnetic microspheres has been used to enrich hematopoietic stem cells from human bone marrow, whole cord blood, or mobilized peripheral blood mononuclear cell collections. This method has been reported to achieve high separation purity of CD34+ cells in small scale experiments with fresh material. The aim of the present study was to compare the efficacy of the CD34+ cell selection technique, when thawed bone marrow or fresh peripheral blood mononuclear cells were enriched. Starting with thawed bone marrow containing 2.9% CD34+ cells the final product purity was 67.7% with a 6% CD34+ cell yield (enrichment factor 25.7), and a 85-fold CFU-GM enrichment. Using fresh mobilized peripheral blood mononuclear cells the released cells contained 77.6% CD34+ cells with a 47% yield (enrichment 86.5-fold), and a 46-fold CFU-GM enrichment. These results indicate that CD34+ cells can be selected from cryopreserved bone marrow using immunomagnetic procedures. However, fresh leukapheresis products seem to be a much better material for a positive immunomagnetic stem cell selection technique in terms of purity, yield and enrichment.     

Umbilical cord blood transplantation: current state of the art

Human leukocyte antigen-matched sibling donor umbilical cord blood transplantation, or 0-2 human leukocyte antigen mismatched unrelated donor umbilical cord blood, is now considered an acceptable alternative to the use of bone marrow as a source for hematopoietic stem cells for pediatric hematopoietic stem cell transplantation, and is being investigated in adults. Major advantages of umbilical cord blood include the speed of availability compared with unrelated donor bone marrow, and tolerance of 1-2 human leukocyte antigen mismatch, which offers the opportunity to extend the donor pool. Umbilical cord blood transplantation is associated with durable engraftment and low incidence of severe graft-versus-host disease, even in the 1-2 human leukocyte antigen mismatched setting. Clinical experience has established the importance of graft cell dose in determining engraftment and survival in unrelated donor umbilical cord blood transplantation. More recently, the influence of human leukocyte antigen on outcome has become apparent. This review outlines the state of the art of umbilical cord blood transplantation, with emphasis on practical considerations in umbilical cord blood selection, and describes current research directions for this hematopoietic stem cell source.     

Use of Flow Cytometric CD34 Analysis to Quantify Hematopoietic Progenitor Cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mono-nuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only l%-9% of the CD34⁺ MNC. Due to the variable numbers of CD34⁺ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA. Exclusion of the CD34⁺/CD45RA⁺⁺ cell fraction will improve the correlation and give results similar to those obtained with untreated blood MNC.     

脐血与成人血成分、T淋巴细胞亚群及正常人骨髓集落形成的比较研究

目的：研究脐血,成人外周血及骨髓的特性,方法：采用全自动血细胞分析仪及流式细胞仪分别测定济血,成人外周血的血成分及T巴细胞亚群,采用集落形成试验观察脐血,外周因及骨髓的CTU－GM和BFU－E的产率变化,结果：脐血的各种血成分均显著高于外周血,CD,CD3明显低于外周血,而CD4,CD8的表达与外周血相似,经细胞因子诱导后,脐血中的CUF0－GM及BFU－E显著升高,结论：脐血富含造血干细胞,是重建造血与免疫系统的血细胞新来源。     

Bone and bone-marrow interactions: haematological activity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide. Mobilizing properties on white blood cells and peripheral blood stem cells in mice.

Osteogenic growth peptide (OGP) increases blood and bone marrow cellularity in mice, and enhances engraftment of bone marrow transplant. Carboxy-terminal pentapeptide (OGP10-14) holds several properties of full-length polypeptide. We evaluated whether synthetic OGP-derived pentapeptide (sOGP10-14) has some activity on peripheral blood cell recovery after cyclophosphamide-induced aplasia, and on stem cell mobilization. Peripheral blood stem cell (PBSC) mobilization was evaluated by administering granulocyte-colony stimulating factor (G-CSF) or sOGP10-14 after cyclophosphamide (CTX) injection. Haematological parameters and CD34/Sca-1 positive cells were sequentially evaluated. Colony-forming tests were performed in bone marrow cells from CTX-, G-CSF- and sOGP10-14-treated mice. sOGP10-14 was able to enhance band cells and monocyte recovery after cyclophosphamide administration. White blood cell (WBC) counts reached the maximum peak by day +10 but, on day +7, a significant recovery was already detected in sOGP10-14 treated mice. On day +10 the WBC increase in sOGP10-14-treated mice was comparable to that found in G-CSF treated ones. Moreover, CD34/Sca-1 positive early precursors were significantly mobilized by sOGP10-14 compared to the control group. In sOGP10-14-treated mice, the colony-forming unit-granulocyte-macrophage-megakaryocyte (GEMM-CFU) and burst-forming unit-erythroid (BFU-E) were significantly increased in bone marrow cells in comparison to mice treated with CTX only. These results suggest a central role of sOGP10-14 in bone and bone marrow interaction, and a possible role of sOGP10-14 as a mobilizing agent.