冻存脐血中血小板、血小板微粒的变化及其与CD34^＋细胞粘附分子表达关系的观察

研究新鲜和冻存脐血中血小板(PLT)、血小板微粒(PMPs)含量的变化，以及它们对脐血CD34^ 造血干／祖细胞粘附分子表达的影响。测定冷冻前后，脐血中PLT、PMPs的含量，以及脐血CD34^ 细胞与PMPs孵育后粘附分子(CD41a，CD61，CD62P)的变化。结果，冻存脐血中PLT含量减少、PMPs含量增加；新鲜PMPs上调CD34^ 细胞粘附分子表达的作用比冻存PMPs强；脐血PLT经过深低温冷冻后仍然能被激发产生PMPs，并能上调CD34^ 细胞粘附分子表达。结果显示：我们可以用冻存的PLT来获得PMPs，以提高干细胞粘附分子表达。     

血小板来源的微粒对脐血CD34+细胞黏附性研究

目的　研究血小板来源的微粒 (PMPs)对人脐血CD34 细胞黏附分子表达的作用效应。方法　采用免疫磁珠法分选出广州市妇婴医院 2 0 0 3年 4～ 6月提供的新鲜采集脐血中的CD34 细胞 ,与PMPs室温孵育15min至 1h。应用流式细胞术测定分选前、分选后以及与PMPs孵育后CD34 细胞的表面黏附分子 (CD4 1a ,CD6 1,CD6 2 p ,CXCR4 )的表达。比较PMPs作用前后CD34 细胞与脐静脉内皮细胞及黏连蛋白的黏附性。结果　 ( 1)CD34 造血干 /祖细胞与PMPs孵育后 ,其黏附分子 (CD4 1a ,CD6 1,CD6 2 p ,CXCR4 )的表达显著增高。( 2 )这些黏附分子表达率有一定变化趋势 :分选后最低 ,全血次之 ,孵育后其表达率达到高峰。 ( 3)与PMPs作用后 ,CD34 细胞的黏附性明显增高 ,同时PMPs不改变CD34 细胞的集落形成能力。结论　PMPs与脐血CD34 细胞结合能增加其黏附性。有可能作为辅助物质应用到脐血干细胞移植当中 ,以促进脐血CD34 细胞的植入     

CD11b对儿童急性白血病脐血移植粒细胞和血小板恢复时间的影响

为了研究CD34+ CD1 1b+ 细胞输入量在脐血移植中对中性粒细胞和血小板恢复时间的影响 ,观察并分析 1 7例儿童急性白血病患者进行无关脐血移植的临床数据。结果显示 ,移植后 1 7例患者中性粒细胞≥ 0 5× 1 0 9 L的时间为 1 1～ 32天 (中位数为 1 7天 ) ;血小板≥ 2 0× 1 0 9 L的时间为 1 2～ 1 1 8天 (中位数为 4 0天 )。CD34+CD1 1b+ 细胞输入量为 1 0 7～ 79 0 0× 1 0 4 Kg(中位数为 9 1 8× 1 0 4 Kg)。CD34+CD1 1b+ 细胞输入量与中性粒细胞恢复时间呈反比 (γ =- 0 4 89,P <0 0 5) ;CD34+CD1 1b+ 细胞输入量与血小板恢复时间无关 (r =- 0 2 38,P >0 0 5)。CD34+ CD1 1b+细胞输入量与造血干细胞移植时的造血重建有关。     

采用二甲基亚砜和右旋糖酐冻存脐血造血细胞的研究

脐血移植已被广泛地用于治疗各种血液性疾病和恶性肿瘤 ,临床上主要采用同胞脐血移植和无关供者脐血移植两种形式[1] 。前者往往直接将整份脐血冻存 ,后者供者来源于脐血库 ,多为经羟乙基淀粉 (ＨＥＳ)分离后的有核细胞(ＮＣ) [2 ] 。对于保存方法 ,单用一种冷冻保护剂即使加大剂量也不会增加冷冻效果 ,反而加大毒性[3 ] 。我们采用合用两种冷冻保护剂冻存整份脐血和分离后有核细胞 ,以探讨理想的冻存方法来建立脐血库和保存同胞脐血。材料和方法选取足月健康新生儿采集脐血。冷冻保护剂甲为 2 0 %二甲基亚砜 (ＤＭＳＯ ,美国Ｓｉｇｍａ公司…     

白细胞相关免疫球蛋白样受体-1在免疫性血小板减少症患儿中的表达

目的 观察白细胞相关免疫球蛋白样受体-1（LAIR-1）在免疫性血小板减少症（ITP）患儿中的表达变化,探讨其在ITP发病中可能的机制。方法 采用流式细胞术检测40例ITP患儿外周血CD4+ T细胞、CD8+ T细胞及CD19+CD20+ B细胞膜LAIR-1的表达率;ELISA检测ITP患儿血清中可溶性LAIR-1（sLAIR-1）含量及实时荧光定量PCR法检测ITP患儿LAIR-1 mRNA表达水平。32名同龄健康儿童作为对照组。结果 ITP组CD19+CD20+ B细胞比例高于对照组（P+ T细胞比例低于对照组（P+ T细胞、CD8+ T细胞膜LAIR-1的表达低于对照组（PP结论 ITP患儿中CD4+ T细胞、CD8+ T细胞膜上LAIR-1表达降低,血清sLAIR-1表达增高,提示LAIR-1可能是导致ITP患儿免疫失衡的重要因素。     

血小板表面TLR2和TLR4在儿童特发性血小板减少性紫癜中的变化及意义

目的：探讨血小板表面Toll样受体TLR2和TLR4、淋巴细胞表面CD86和血清中IL-2,IFN-γ,IL-4,IL-10表达的变化及相关性在儿童特发性血小板减少性紫癜（ITP）发病中的意义。方法：流式细胞学技术检测24例急性特发性血小板减少性紫癜(AITP)患儿、21例慢性特发性血小板减少性紫癜（CITP）患儿和20例对照组儿童血小板表面TLR2和TLR4,CD86的表达水平;ABC-ELISA法检测血清中IL-2,IL-4,IL-10和IFN-γ的浓度。结果：①AITP组、CITP组患儿CD41+TLR2+和CD61+TLR4+表达显著低于对照组(P<0.01);且AITP组显著低于CITP组（P<0.01);②AITP组和CITP组CD86+表达较对照组显著增高（P<0.01）,AITP组和CITP组间差异无显著性;③AITP组和CITP组血清IL-2,IL-4,IL-10及IFN-γ浓度均较对照组显著增高（P＜0.05）,AITP组和CITP组间差异无显著性。④相关性分析：CD41+TLR2+表达与CD61+TLR4+表达呈正相关,二者均与CD86+表达及血清IL-2,IL-4,IL-10浓度呈负相关,与血清IFN-γ浓度无相关性。结论：联合检测血小板表面TLR2和TLR4、淋巴细胞表面CD86的表达和血清中IL-2,IFN-γ,IL-4,IL-10含量的变化及相关性对于研究儿童ITP的发病机制以及早期分型具有重要参考价值。［中国当代儿科杂志,2009,11（10）：797-801］     

干扰素治疗特发性血小板减少性紫癜患儿T细胞亚群的变化及临床意义

为探讨T细胞亚群在特发性血小板减少性紫癜 (ITP)中的变化及其用干扰素 (IFN)治疗的影响。采用IFN治疗ITP患儿 ,分别于治疗前后用SAP法检测T细胞亚群的含量。结果 ,ITP患儿IFN治疗前外周血CD4 降低 ,CD8升高 ,CD4 CD8显著降低 ;IFN治疗后CD4 ,CD8降低 ,CD4 CD8明显高于治疗前。结果表明 ,ITP患儿T细胞亚群表达及比例失调 ,IFN治疗ITP有显著临床疗效     

CD34+CXCR4+与非亲缘脐血移植后粒细胞和血小板重建关系的研究

目的　研究回输CD34+CXCR4 +的输入量在非亲缘脐血移植治疗儿童急性白血病中对中性粒细胞(ANC)和血小板 (BPC)恢复时间的影响。方法　用流式细胞术分析回输CD34+CXCR4 +细胞数 ,并对 2 3例儿童急性白血病在非亲缘脐血移植后的ANC和BPC恢复时间等临床资料进行测定。结果　 2 3例患儿中 ,CD34+CXCR4 +输入量为 (2 1～ 198 6 )× 10 4/kg(中位数 19 9× 10 4/kg)。在 2 1例植入成功患儿中 ,ANC >0 5× 10 9/L的时间为 11～ 4 1d(中位数 2 0d) ;而在 2 0例获得统计资料的患儿中 ,BPC >2 0× 10 9/L的时间为 12～ 12 0d(中位数4 4 5d)。CD34+细胞输入量与ANC、BPC恢复时间的r值分别为 - 0 35 4 (P =0 116 )、- 0 4 30 (P =0 0 5 8) ,CD34+CXCR4 +细胞输入量与ANC、BPC恢复时间的r值分别为 - 0 5 2 7(P =0 0 17)、- 0 6 2 9(P =0 0 0 4 )。结论　CD34+CXCR4 +输入量与非亲缘脐血移植后的造血重建有关。     

特发性血小板减少性紫癜儿童外周血调节性T细胞的变化

目的：研究特发性血小板减少性紫癜(ITP)患儿外周血CD4+CD25+CD127-及CD3+CD4-CD8-调节性T细胞（Treg）的变化及意义。方法：采用免疫荧光流式细胞技术检测33例ITP患儿及21例正常儿童外周血CD4+CD25+CD127-及CD3+CD4-CD8- Treg水平。结果：ITP患儿CD4+CD25+CD127-及CD3+CD4-CD8- Treg百分比明显低于正常儿童,分别为(2.7±1.7)％ vs (4.8±1.6)％;(5.2±3.1)％vs (8.1±3.5)％(P<0.01)。结论：ITP患儿CD4+CD25+CD127-及CD3+CD4-CD8- Treg百分率降低,提示其可能参与了ITP的发病机制。     

脐血和外周血来源的巨核细胞体外扩增差异的研究

目的建立外周血(peripheral blood,PB)和脐血(cord blood,CB)来源的CD3+4细胞体外定向诱导扩增巨核细胞(megakaryocyte,MK)的最佳体系,探讨两种来源巨核细胞的扩增差异。方法以Ficoll-Hapaque分离“动员”的外周血和脐血单个核细胞,免疫磁珠分离纯化CD3+4细胞,在含胎牛血清的液体培养体系中,以不同细胞因子组合诱导两种来源的CD3+4细胞,定时进行细胞计数和流式细胞术检测培养体系中CD4+1细胞的含量;同时采用甲基纤维素半固体培养法进行巨核细胞集落培养,测定巨核细胞集落形成单位的数量。结果在血小板生成素(thrombopoietin,TPO)+胎肝酪氨酸激酶配体(FLT-3ligand,FL)+白介素6(interleukin-6,IL-6)+IL-3组合中,外周血来源的CD4+1细胞第10天扩增了131±18倍,脐血来源的CD4+1细胞在培养的第14天扩增了193±25倍,为增殖高峰。均明显高于同来源的其他3组(P<0.05),随着时间的推移两者的CD4+1细胞扩增倍数均呈下降趋势。结论TPO+FL+IL-6+IL-3组合均为CB和PB体外诱导扩增巨核细胞的最佳组合。CB来源的巨核细胞较PB来源的巨核细胞有更强的增生能力,而PB来源的CD3+4细胞产生巨核细胞的时间较CB来源的短,与临床上外周血造血干细胞移植的血小板造血重建快于脐血移植相一致。     

The in vitro effects of stem cell factor and PIXY321 on myeloid progenitor formation (CFU-GM) from immunomagnetic separated CD34+ cord blood.

Two novel cytokines, stem cell factor (SCF) and PIXY321 (a fusion protein, granulocyte macrophage colony-stimulating factor+IL-3), have recently been demonstrated to enhance in vitro adult myelopoiesis. In this study, we compared the success of separating very early hematopoietic progenitor cells (CD34+) from both cord blood (CB) and adult bone marrow (ABM) and their differential response to SCF, PIXY321, and other later-acting colony-stimulating factors (CSF). Briefly, CD34+ cells were isolated from CB and ABM with an anti-CD34 MAb, HPCA-1, and incubated with various combinations of SCF, PIXY321, and other CSF. The percentage of CD34+ cells was decreased in CB compared to ABM before separation (0.54 versus 1.71%) (p = 0.05). Isolated CD34+ cells from CB and ABM were similar in lineage with respect to CD38, HLA-DR, CD33, and CD5, but decreased in CB with respect to B-lineage expression (CD19, CD10, and CD22) (p = 0.05). SCF increased colony forming unit-granulocyte-macrophage (CFU-GM) formation from CB CD34+ cells compared to unconditioned media and had a significant additive increase with IL-3 (p = 0.006) and granulocyte colony-stimulating factor (p = 0.03). SCF also had an additive increase in CB CFU-GM formation with PIXY321 (p = 0.007). PIXY321 had a similar increase in CFU-GM formation from both CB and ABM CD34+ cells compared to the combination granulocyte macrophage colony-stimulating factor + IL-3. When SCF was added to IL-3, PIXY321, or PIXY321 + IL-6, there was an increase in CFU-GM from CB versus ABM CD34+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

二甲基亚砜和右旋糖酐应用于脐血造血细胞的低温保存

探讨液氮保存中联合低温保护剂 (二甲基亚砜和右旋糖酐 )对造血细胞的保护作用和不同冻存时间对造血细胞活力的影响。同时分析脐血全血 4℃保存造血细胞的变化。通过测定冻存前后总有核细胞数 ( TNC)、细胞活率、造血祖细胞集落生成情况 ,反映冻存后造血细胞损失情况及增殖能力。结果显示 40例脐血冻存后细胞活率平均为 91 .5 % ,TNC、CFU- GM、BFU- E、CFU- GEMM平均回收率为 95 .0 %、86 .7%、81 .8%、84.4%。不同保存时间细胞仍保持较高细胞活率和祖细胞产率 ,48小时细胞活率和祖细胞产率有明显下降。二甲基亚砜 ( DMSO)与右旋糖酐 ( Dextran)组成的联合保护剂对造血细胞有良好保护作用 ,适用于造血细胞的长期低温保存。长期冻存造血细胞的效果与冻存时间无密切关系。全血 4℃保存应在 2 4小时分离冻存。     

TPO和FL有效扩增脐血CD34+细胞为巨核前体细胞

目的探讨人脐血CD34+细胞在TPO和FL细胞因子组合作用下体外扩增为巨核前体细胞的效果,为开展血小板减少症的细胞治疗建立实验条件。方法本研究采用人脐血经免疫磁珠法纯化分离的CD34+细胞,研究TPO+FL细胞因子组合对体外巨核前体细胞的扩增效果。结果显示在TPO与FL组合的作用下,脐血CD34+细胞(n=6)显著地扩增为巨核前体细胞(CD61+CD41+细胞),作用14天为较理想时间。无论是全细胞数和CD34+细胞,或是CD61+CD41+细胞和CFUMK,经过体外扩增,均显著增加。结论研究表明脐血CD34+细胞在TPO和FL细胞因子组合作用下可有效地扩增为巨核前体细胞,第14天是培养较理想时间。     

脐血巨核细胞体外扩增及其功能的研究

目的研究扩增后脐血巨核细胞的生物学特性和功能,为脐血巨核细胞的扩增和临床应用提供依据。方法收集足月妊娠健康新生儿的脐带血,免疫磁珠法分离出其中的CD34^＋细胞。采用血小板生长因子（thrombopoietin,TPO）＋干细胞因子（stem cell factor,SCF）＋白细胞介素3（interleukin-3,IL-3）＋IL-6和TPO＋SCF两种细胞因子组合,将富集的脐血CD34^＋接种于无血清无基质细胞的悬浮培养体系中,分别在3、7、10、14d收集扩增产物。运用流式细胞术检测巨核细胞的表型;血浆块法检测巨核细胞集落（colony forming unit-megakaryocyte,CFU-MK）的形成;对巨核细胞进行DNA含量检测以评价其成熟程度;血小板体外活化实验及SCID小鼠体内移植实验评价扩增后巨核细胞的功能。结果不同细胞因子组合和培养时间扩增后,巨核细胞的数量和生物学特性不同。随着培养时间的延长,巨核细胞（CD41^＋）的数量逐渐增加,但培养至14d时增势减缓。因子组合TPO＋SCF＋IL-3＋IL-6各时间段的扩增能力（分别扩增5．2、40．7、121．2、149．7倍）均比因子组合TPO＋SCF（分别扩增3．8、27．4、85．9、106．5倍）强,但因子组合TPO＋SCF的扩增能力仍能满足临床的需要。巨核祖细胞（CD34^＋CD41^＋）的数量在第7天时最多（分别增加43．4和36．2倍）,这也被CFU-Mk所证实。DNA含量检测发现,随着培养天数的增加,多倍体细胞所占的百分比增加。体外血小板活化实验证实,扩增的巨核细胞在体外可产生血小板,有正常巨核细胞功能。移植后两组小鼠的骨髓中均检测到人CD45^＋和CD41^＋细胞。小鼠外周血中人血小板在移植后3d就可测到,5d就可达到高水平（分别为20．7％和17．9％）,维持20d以后才逐渐下降。结论通过对扩增后巨核细胞的生物学特性的研究,有助于寻找有效、简便、易于植入受者体内的扩增方法。体外扩增的脐血巨核细胞可植入骨髓并产生功能正常的血小板。     

Analysis and characterization of hematopoietic progenitor cells from fetal bone marrow, adult bone marrow, peripheral blood, and cord blood.

Hematopoietic stem cell transplantation has been increasingly used to replace a defective hematopoietic system and to treat various genetic defects as well as malignant diseases. However, the limitations of conventional bone marrow transplantation have stimulated an intense interest in exploring the use of alternative sources of hematopoietic stem cells, including peripheral blood mononuclear cells (PBMC) and cord blood (CB). A major investigative effort of our laboratory has been focused on evaluating fetal bone marrow (FBM) for transplantation. The current study compares and characterizes the functional and phenotypic characteristics of FBM, CB, adult bone marrow (ABM), and PBMC by clonogenicity assays, immunogenicity, and the quantification of progenitor cells. There was a striking difference in the proportion of CD34+ cells in FBM, ABM, PBMC, and CB (24.6%, 2.1%, 0.5%, and 2.0%, respectively). The clonogenic potential, as measured by colony forming unit in culture (CFU-C) assay, was significantly higher in FBM when compared with ABM, PBMC, and CB (202.5, 73.5, 40.8, and 65.5 colonies/10(5) cells, respectively). There was a significant decrease in proliferative responsiveness in mixed lymphocyte reaction (MLR) assay of FBM and CB compared with ABM and PBMC. These observations indicate that each source of hematopoietic stem cells has different intrinsic properties closely correlated with ontogenetic age that is a vital determinant for phenotypic characteristics, lineage commitments, immunogenicity, and proliferative potentials.     

脐血造血细胞含量与白血病脐血移植疗效分析

目的分析4711份库存脐血造血细胞含量及探讨脐血造血细胞含量与白血病脐血移植疗效的关系。方法分析4711例库存脐血总有核细胞数(TNC)和CD34+细胞数分布情况,探讨不同的造血细胞输入量、供受者HLA不相合数、受者性别、年龄、体重和疾病类型间植入率和生存率的差异。结果 4711例库存脐血TNC和CD34+细胞中位数分别为1.14×109/kg和4.06×106/kg,按3.7×107/kg有效TNC输入量计算,93.2%脐血可供体重50 kg以下受者移植。89例白血病患者移植后植入75例,植入率为84.3%。中性粒细胞绝对值≥0.5×109/L、血小板≥20×109/L和≥50×109/L的时间分别为移植后17、34和46 d。75例植入病例中,长期无病存活47例,死亡26例,2例复发;急性移植物抗宿主病(GVHD)Ⅰ～Ⅱ度、Ⅲ～Ⅳ度和慢性GVHD发生率分别为54.7%、20.0%、9.3%。影响移植植入率的因素包括受者年龄、TNC和CD34+细胞输入量;影响生存率的因素包括受者年龄、体重和输入CD34+细胞数。结论在无法找到HLA全相合骨髓供者时,可选择脐血作为替代骨髓的造血干细胞来源治疗儿童与成人白血病,TNC和CD34+细胞数仍是选择脐血移植物的参考指标。     

脐带血造血干细胞的4℃保存与移植

为建立一种简便而有效的脐带血造血干细胞低温保存方法，我们探讨了4℃保存脐带血并用于临床移植的可行性。结果显示：脐带血4℃低温保存2～3天，造血干细胞回收率基本稳定；应用4℃保存46小时的脐带血（有核细胞数1．05×108／kg体重）移植治疗1例急性淋巴细胞白血病患儿，获得骨髓造血功能重建，有明确植活证据，并使白血病癌基因消失。说明脐带血的4℃保存具有一定临床价值。     

Immunophenotypic changes of fetal cord blood hematopoietic progenitor cells during gestation

We measured cell surface expression of CD34, HLA-DR, CD38, CD19, CD33, CD71, and CD45 antigens in the hematopoietic progenitor cells of fetal cord blood to investigate immunophenotypic changes at different gestational ages. These antigens were identified by flow cytometry in 11 fetuses (gestational age 19-24 wk, in 12 preterm (25-28 wk) and in ten newborn infants born at term. The frequency and number of CD34+ cells were higher in the blood of the 11 fetuses; in addition, a statistically significant inverse correlation between number of CD34+ cells and advancing gestational age was noted. The numbers of CD34+ CD19+, CD34+ CD33+, and CD34+ CD45+ coexpressing cells were significantly higher in the fetuses, whereas CD34+ CD38+ cells were more represented in the neonates at term. Gestational age was inversely correlated with the number of CD34+ CD19+ and CD34+ CD33+ coexpressing cells. A positive correlation between gestational age and CD34+ CD38+ cells was noted. The number of CD34- CD19+, CD34- CD38+, and CD34- CD45+ cells was higher in term infants; furthermore, a significant correlation between advancing gestational age and CD34- CD38+ or CD34- CD45+ cells was demonstrated. The proliferative capacity was also higher at lower gestational ages. These data suggest that the development and lineage commitment of fetal cord blood hematopoietic progenitor cells are very active during the last two trimesters of pregnancy. The most significant changes of hematopoietic cells maturation seem to occur within 25 wk of gestation.     

Intrabone cord blood hematopoietic stem cell transplantation in a subset of very high-risk pediatric patients: a safety and feasibility pilot study

The main limit of umbilical cord blood hematopoietic stem cell transplantation is a more difficult engraftment related to the number of cells infused per kilogram of recipient body weight. This limit makes the cord blood a suboptimal source of hematopoietic stem cells for transplantation in case of difficult engraftment situations. Direct intrabone cord blood (CB) injection has been recently investigated as a solution to cell dose problem in the adults population, but there is a lack of data concerning this approach in pediatric patients. Here, we describe 5 pediatric patients undergoing intrabone cord blood transplantation (IBCBT) for different diseases characterized by a high risk of posttransplant graft failure. The conditioning regimen differed according to the disease, whereas the GvHD prophylaxis consisted of cyclosporine, mycophenolate, and ATG. The median numbers of total nucleated cells infused and CD34(+) cells were 3.3 × 10(7)/kg, 2 × 10(5)/kg. All the patients showed complete hematological recovery and complete donor engraftment. No patient had secondary graft failure, whereas 1 patient relapsed 6 months after IBCBT. No patient died of transplant-related complications. Our results show that IBCBT is safe and feasible in pediatrics as well, and suggest that IBCBT might be an attractive option to overcome some limits of umbilical cord blood hematopoietic stem cell transplantation.