人胎盘组织来源造血干/祖细胞及其特性

背景:近年来的研究表明,除已知的人骨髓、外周血和脐带血中存在造血干/祖细胞外,人胎盘组织中也有造血干/祖细胞存在.目前为止,还缺乏对人胎盘组织造血干/祖细胞的增殖分化特性及人胎盘组织淋巴细胞亚群组成和免疫原性等的深入研究.目的:探究人胎盘组织是否含有比脐带血更丰富的造血干/祖细胞,并对其造血祖细胞系增殖分化能力进行检测,同时对人胎盘组织淋巴细胞亚群组成及表型特征进行分析.设计、时间及地点:开放性实验,于2004-01/2006-12在贵州省细胞工程重点实验室完成.材料:经产妇知情同意,无菌采集遵义医学院附属医院产科健康足月分娩新生儿胎盘和脐带血共12份.淋巴细胞亚群检测试剂盒,CD34绝对计数试剂盒(Becton Dickinson公司):CD34磁珠分选试剂盒,FITC标记的CD38单克隆抗体,抗FITC磁珠和MS/LS免疫磁式细胞分选柱(Miltenyi Biotec).方法:脐带血与RPMI-1640培养基(含体积分数为0.1的胎牛血清)按1:1的比例混合,采用Ficoll-Histopaque分离液离心30min,吸取界面层细胞,PBS洗涤一次,获得脐带血单个核细胞.采用机械法加0.25g/L胶原酶消化制备胎盘组织单个细胞悬液,之后同脐带血单个核细胞分离步骤分离胎盘单个核细胞.流式细胞仪检测胎盘单个核细胞中CD34 CD38-, CD34 CD38 造血干/祖细胞(HSPCs)和淋巴细胞亚群的组成比例.免疫磁珠分选法分选人胎盘CD34 CD38-,CD34 D38 造血干/祖细胞,并分别进行粒细胞-单核细胞集落生成单位、红细胞爆裂型集落生成单位、混合集落生成单位系集落形成培养,以评价其造血祖细胞系增殖分化能力.实验全程用脐带血作平行比较分析.主要观察指标:胎盘和脐带血CD34 造血干/祖细胞组成百分率、祖细胞系集落形成能力、淋巴细胞亚群表型及组成特点.结果:[1]胎盘CD34 造血干/祖细胞百分率是脐带血的8.8倍,差异有显著性意义(P<0.01).[2]胎盘中的淋巴细胞总数、T细胞(CD3 CD2 )、B细胞(CD19 )、Th(CD3 CD4 )细胞及Th/Ts比值均明显低于脐带血,而CD8 CD28-T抑制细胞则明显高于脐带血,差异有显著性意义(P<0.01).[3]胎盘CD34 CD38 造血干/祖细胞亚群培养形成的粒细胞-单核细胞集落生成单位、红细胞爆裂型集落生成单位、混合集落生成单位集落数明显高于CD34 CD38-造血干/祖细胞亚群(P<0.01);胎盘与脐带血造血干/祖细胞中相同表型细胞亚群形成的各系集落数比较,差异无显著性意义(P0.05).结论:人胎盘组织富含CD34 造血干/祖细胞,其CD34 CD38 、CD34 CD38-两个造血干/祖细胞亚群均具有增殖分化为粒细胞-单核细胞集落生成单位、红细胞爆裂型集落生成单位、混合集落生成单位的能力,并且人胎盘组织具有淋巴细胞亚群低比例和抑制性T细胞高比例的特点,使其有望成为造血干/祖细胞移植的新来源.     

Cesarean section due to fetal distress increases the number of stem cells in umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) can be used as hematopoietic stem cell source for transplantation. The success of a transplantation is highly correlated with the number of total nucleated cells (TNCs) and CD34+ cells in the UCB. Certain obstetric factors increase the yield of stem cells in the UCB. It is necessary to evaluate optimal conditions in labor to decrease the rate of sample rejection due to low cell count. No data exist regarding the difference between primary and secondary Cesarean sections in terms of efficacy of stem cell harvesting. STUDY DESIGN AND METHODS: Seventy-nine consecutive UCB units from women who had a Cesarean section between 1997 and 2003 were included. The number of TNCs, CD34+ cells, colony-forming units (CFUs), white blood cells (WBCs), nucleated red blood cells (NRBCs), and the total collection volume were compared between cases with primary and secondary Cesarean section. RESULTS: UCB obtained after a Cesarean section due to fetal distress has significantly higher numbers of TNCs, CD34+ cells, NRBCs, and WBCs compared to elective Cesarean section. Of the cases with secondary Cesarean section due to fetal distress, 67 percent resulted in UCB units with sufficient TNC numbers (> or =80 x 10(7) TNCs) compared to 42 percent of the cases with primary Cesarean section. CONCLUSION: Fetal distress increases the number of hematopoietic stem cells mobilized into UCB. Particular effort should be made to collect UCB from newborns who experienced fetal distress.     

不同时相移植人骨髓间充质干细胞对人脐血CD34^＋细胞植入NOD／SCID小鼠的影响

为了观察不同时相移植人骨髓间充质干细胞（MSC）对脐血（UCB）CD34^＋细胞移植的NOD／SCID小鼠造血重建的影响，明确最佳的移植时机，将体外培养扩增的人骨髓MSC分别于UCBCD34^＋细胞移植同时、移植前48小时及移植后48小时输入经^60Coγ射线照射的NOD／SCID小鼠，观察共移植后42天内小鼠外周血白细胞和血小板变化，并于移植后42天处死小鼠，用FACS检测外周血、骨髓和脾脏人源细胞含量。结果表明：（1）MSC和UCBCD34^＋细胞同时输注可明显降低外周血白细胞和血小板下降幅度，缩短白细胞和血小板恢复时间；二者不同时输注均不降低白细胞和血小板下降幅度，且输注UCBCD34^＋细胞后48小时输注MSC时外周血血小板恢复时间明显晚于同时输注者。（2）与单纯UCBCD34^＋细胞移植相比较，不同时相输注MSC均可促进UCBCD34^＋细胞的植入，三个共输注组间促进骨髓各系造血植入效应无明显差异。结论：人骨髓MSC与UCBCD34^＋细胞共移植时，以同时移植效果最佳，此结果为MSC的临床应用提供了实验依据。     

双份脐血间CD34^＋细胞与CD3^＋细胞相互作用对分化增殖能力的影响

目的双份脐血移植的植入动力学机制目前尚无定论,推测双份脐血中的淋巴细胞与优势份脐血的产生相关。本实验将双份脐血的CD34^＋细胞与CD3^＋细胞混合培养,观察CD3^＋细胞对CD34^＋细胞的增殖分化有无影响。方法建立液体和半固体培养体系,将免疫磁珠分选纯化的双份脐血间的CD34^＋细胞和CD3^＋细胞混合培养6d和14d。以流式细胞计数观测CD34^＋细胞培养后的分化指标（CD33,CD41,CD71）;计数集落形成单位（GM—CFU、BFU-E、GEMM—CFU）分析CD34^＋细胞的增殖情况。结果液体共培养后各份CD34^＋细胞表面分化指标的变化。脐血CD34^＋细胞分选富集的纯度为（98．70±0．72）％。3d实验组和对照组的各项分化指标无差异（P〉0．05）;6d的CD33、CD71实验组明显低于对照组,而CD41明显高于对照组（P〈0．05）。半固体共培养后CD34^＋细胞增殖能力的变化。实验组的红系集落形成单位（BFU—E）及粒单细胞集落形成单位（GM—CFU）数低于对照组（P〈0．05）,而混合细胞集落形成数（GEMM—CFU）高于对照组（P〈0．05）。结论将两份脐血的CD34^＋细胞和CD3^＋细胞体外混合培养对CD34^＋细胞的增殖分化能力有影响,推测双份脐血间的相互作用可部分地通过CD3^＋细胞介导。     

Efficient adenoviral vector transduction of human hematopoietic SCID-repopulating and long-term culture-initiating cells

This article presents our studies on the adenoviral transduction efficiency, level of transgene expression, cell cycle status, and multilineage reconstitution ability of human CD34+ hematopoietic cells transduced under proliferating and survival growth conditions. Bone marrow and umbilical cord blood CD34+ cells were cultured in serum-free medium under survival conditions with thrombopoietin (Tpo) alone, or under proliferating conditions with Tpo, c-Kit ligand (KL), and Flt3 ligand (FL). Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of the PGK-1 promoter were used to transduce CD34+ cells. Approximately 10% of CD34+ cells were EGFP+ under both culture conditions. In contrast, up to 50% of CD34+CD38- cells were EGFP+, whereas a maximum of 8% of CD34+CD38(high) cells were EGFP+ (p < 0.001). Both colony-forming unit cells (CFU-C) and 5-week long-term culture-initiating cells (LTC-ICs) were efficiently transduced. Under survival conditions, a substantial fraction of transduced CD34+ cells remained quiescent. The nondividing CD34+EGFP+ cells contained LTC-ICs capable of reconstituting longterm culture for as long as 10 weeks. CD34+EGFP+ cells also retained the ability to engraft and multilineage-reconstitute NOD/SCID mice. These observations demonstrate that primitive human hematopoietic progenitor cells can be efficiently transduced by adenoviral vectors.     

脐血造血干/祖细胞免疫表型的流式细胞术双标法分析及其意义

目的比较脐血和骨髓中造血干/祖细胞(HSPC)的免疫表型差异.方法使用流式细胞术(FCM)双标法对38份脐血及10份骨髓HSPC进行免疫表型分析.结果①脐血有核细胞中CD34+细胞所占比例与骨髓中相近,约为0.5%;②脐血CD34+细胞中CD34+CD38-[(17.C4±5.37)%]、CD34+HLA-DR-[(32.65±10.71)%]及CD34+H-CAM+(CD44+)[(77.84±7.69)%]亚群含量均高于骨髓[含量分别为(8.26±3.19)%、(14.05±1.67)%和(70.02±6.40)%],CD34+CD13+、CD34+CD19+亚群比例低于骨髓.结论脐血与骨髓CD34+细胞比例相近,但前者较原始的干细胞含量更高,故脐血是极具潜力的HSPC来源;而脐血CD34+细胞中髓系及淋系祖细胞含量低于骨髓,可能是脐血移植后造血及免疫重建缓慢的原因之一.     

体外扩增脐血CD34+细胞的实验研究

目的 探讨体外扩增脐血干／祖细胞用于成人脐血移植的可能性。方法 从１０份新鲜的脐血标本中纯化的ＣＤ３４＾＋细胞接种于含体积分数为２０％的胎牛血清（ＦＢＳ）的ＩＭＤＭ培养基的悬 培养体系中,分别加入由ＳＣＦ、Ｆｌｔ－３Ｌｉｇａｎｄ（ＦＬ）与ＩＬ－１β、ＩＬ－３、ＩＬ－６、Ｇ－ＣＳＦ、Ｅｐｏ（合称１３６ＧＥ）组成的３组细胞因子（Ａ组：１３６ＧＥ＋ＦＬ;Ｂ组：ＳＣＦ＋１３６ＧＥ;Ｃ组：ＦＬ＋ＳＣＦ＋１３     

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots

BACKGROUND: The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability.
STUDY DESIGN AND METHODS: The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration.
RESULTS: Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 ± 0.22 percent, the results after thawing were 0.98 ± 0.65 and 0.57 ± 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition.
CONCLUSIONS: The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.     

Assessment of cell viability and apoptosis in human umbilical cord blood following storage

There is increasing use of human umbilical cord blood (UCB) stem cells for unrelated donor transplantation. Successful engraftment depends in large measure on the dose and quality of cells in the UCB unit. In the present study, we attempted to identify a simple and rapid technique for assessing the quality and recovery of UCB cells following laboratory manipulation. Mononuclear cells (MNC) from fresh (<48 h old) and thawed UCB units were stained with 7-amino-actinomycin D (7-AAD), revealing 2-3% dead cells. The frequencies of apoptotic cells in fresh and thawed sample were similar. However, UCB held for 72 h showed higher levels of cell deterioration. Interestingly, staining with 7-AAD was more sensitive to cellular damage than was uptake of Trypan Blue. 7-AAD staining of MNC also correlated with retention of hematopoietic function (progenitor assays) such that 7-AAD staining frequencies <20% predicted maintenance of hematopoietic cells. Importantly, hematopoietic precursors were less susceptible to storage injury than were UCB MNC as a whole. MNC showed higher levels of 7-AAD staining and apoptosis than did CD34(+) cells. This observation was confirmed in studies of caspase-3 activation, where MNC consistently showed higher frequencies of activation than did CD34(+) cells, especially after overnight storage. Furthermore, antiapoptotic proteins Bcl-2 and Bcl-x were expressed more consistently in CD34(+) cells than in total MNC. In contrast, Bax levels increased with MNC apoptosis. In conclusion, the data suggest that 7-AAD staining of UCB MNC provides a rapid and simple technique for assessing the viability, recovery, and hematopoietic functionality of stored UCB.     

Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.     

间充质干细胞与人脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响

目的 观察间充质干细胞(MSC)与不同比例脐血CD34+细胞共移植对NOD/SCID小鼠造血重建的影响,明确MSC与脐血CD34+细胞共移植的最适数量.方法 给60Coγ射线照射的雌性NOD/SCID小鼠共移植人MSC和不同比例的脐血CD34+细胞,观察共移植后42 d内小鼠外周血白细胞和血小板变化,并于移植后42 d处死小鼠,用流式细胞术检测外周血、骨髓和脾脏人源细胞含量.结果 与单纯脐血CD34+细胞移植相比较:①脐血CD34+细胞与1、5和10倍数量的MSC共移植时,可明显减轻外周血白细胞和皿小板的下降幅度(P<0.01),提前1周使白细胞和血小板恢复至正常水平(P<0.05),三组间差异无统计学意义(P>0.05);②MSC与不同比例的脐血CD34+细胞共移植均可明显提高外周血、骨髓和脾脏造血细胞植入率.比例为10:1时,外周血、骨髓和脾脏中的人源细胞(huCD45+细胞)含量分别增加了(2.8±0.6)倍、(3.5±0.9)倍和(5.2±0.6)倍,增加倍数差异均有统计学意义(P<0.01),达到了最佳的植入效果.结论 脐血CD34+细胞与10倍数量的MSC共移植可达到最佳的促进造血重建作用.     

胎盘组织及血液中含有丰富的造血干/祖细胞

大量的临床移植表明人脐血 (UCB)可以在造血干细胞移植的儿童中得到造血重建 ,可是在成人中脐血移植 (UCBT)效果并不理想 ,这主要是脐血中所含的细胞数及造血干 /祖细胞数有限 ,而不适宜体重较大的成人。本研究在收集脐血的同时 ,也分别收集胎盘血 (UPB)及胎盘组织 (UPT)中的细胞 ,检测有核细胞数 ,CD34(造血干 /祖细胞的表明标记 )阳性细胞 ,粒单细胞集落 (CFU GM)。结果发现 :来自于胎盘血和胎盘组织中的有核细胞数是脐血细胞的 3- 4倍 ;脐血、胎盘血及胎盘组织细胞的有核细胞数分别为 (8.3± 1.0 4 )× 10 8,(16 .33± 5 .5 4 )× 10 8和(8.0 1± 2 .6 4 )× 10 8;CD34+ 细胞分别为 (0 .77± 0 .0 1)× 10 6,(1.2 5± 0 .5 5 )× 10 6和 (4.2 1± 1.90 )× 10 6;在细胞的长期培养中 ,UPB细胞和UPT细胞能生存更长时间 ,而且这些细胞更易贴壁形成纤维样的细胞 ;冰冻前后UPT细胞活性及回收率无明显差异 ,表明胎盘细胞和脐血细胞一样适合于长期储存 ;而且 ,在UPB和UPT中含有更高比例的T淋巴细胞抑制细胞群 ,可能意味着UPB和UPT具有更强的免疫抑制功能。结论指出 ,有必要建立含有胎盘和脐血细胞的血库 ,以便为大剂量放疗及化疗后的儿童及成年病人进行造血干细胞移植 ,为重建造血创造条件     

A large number of mature and functional dendritic cells can be efficiently generated from umbilical cord blood–derived mononuclear cells by a simple two‐step culture method

BACKGROUND: Advances in the past two decades in dendritic cell (DC) biology paved the way to exploit them as a promising tool in cancer immunotherapy. The prerequisite for DC vaccine preparations is large‐scale in vitro generations of homogeneous, mature, and functional DCs. Frequent improvements are being made in the existing in vitro DC production protocols to achieve this goal. In our previous study we reported a large‐scale generation of mature, functional DCs from umbilical cord blood (UCB) CD34+ cells. Here we report that this method can be used for the efficient generation of DCs from UCB mononuclear cells (MNCs) and thus the hematopoietic stem cell isolation step is not essential. STUDY DESIGN AND METHODS: MNCs or CD34+ cells isolated from the same cord blood (CB) samples were used for the generation of DCs. DCs were characterized for morphology, phenotype, and functional assays including antigen uptake, chemotaxis, and mixed leukocyte reaction. Similarly DCs generated from the MNCs of same fresh and frozen CB units were compared. RESULTS: The morphologic, phenotypic, and functional characterization of the DCs generated from various sets show that they were comparable in nature irrespective of the starting population used. CONCLUSION: We conclude that the CD34+ isolation step is not essential for the generation of mature, functional DCs and thus can be eliminated. More importantly, we show that DCs can be generated with equal efficiency from the MNCs of frozen CB units. Our culture method will be useful for exploiting the potential of UCB as an additional source for allogeneic DCs in the clinical settings.     

气相液氮罐内温度差异对脐带血造血干细胞保存质量的影响

目的 研究气相液氮罐内因位置不同而导致的温度差异对脐带血造血干细胞保存质量的影响.方法 选取位于液氮中的脐血干细胞20份作为对照组A组,保存在气相液氮罐内最低温度保存处的20份样本为实验B组,最高温度保存处的20组样品为实验C组.各组细胞解冻复苏后进行有核细胞计数、有核细胞活性、CD34+％、CFU-GM等检测,进行单...     

骨髓基质细胞联合细胞因子对脐血单个核细胞表面抗原表达的影响

背景:课题组已建立胎儿骨髓基质细胞联合细胞因子的造血细胞体外培养体系,该培养体系能否有效扩增各个发育阶段的造血细胞有待验证。目的:观察骨髓基质细胞联合细胞因子培养体系对脐血单个核细胞表面抗原CD133、CD34表达的影响。方法:将从脐血标本中分离出来的单个核细胞接种于无血清培养体系,实验分为3组:①F组:干细胞因子+Flt3配体+促血小板生成素+单个核细胞。②S组:基质细胞+单个核细胞。③SF组:基质细胞+干细胞因子+Flt3配体+促血小板生成素+单个核细胞。在第0,6,10,14天检测有核细胞总数、CD133+、CD34+、CD133+CD34+细胞数以及集落形成单位数。结果与结论:SF组有核细胞总数在各个检测时间点均比其他两组高;除了第14天外,第6、10天两个时间点SF组中CD133+、CD34+、CD133+CD34+细胞及集落形成单位数均高于其他组;含骨髓基质细胞的S组和SF组中CD133+细胞/有核细胞、CD34+细胞/有核细胞、CD133+CD34+细胞/有核细胞的比例保持在较高的水平。结果说明骨髓基质细胞联合细胞因子能有效的扩增脐血单个核细胞及其中的CD133+、CD34+、CD133+CD34+细胞,基质细胞对维持造血干细胞的原始性具有重要的作用。     

不同细胞因子组合对人脐血单个核细胞体外扩增及CD49d和CXCR4表达的影响

为了探讨不同的细胞因子组合对脐血单个核细胞体外的扩增作用及扩增后CD49d和CXCR4的变化，将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7天，在0天，7天检测有核细胞数，CD34^＋细胞数及CD34^＋CXCR4^＋，CD34^＋CD49d^＋的细胞数和集落形成单位（CFU）数．根据不同细胞因子组合实验分组为：对照组；SF组（SCF＋FL）；SFT组（SCF＋FL＋TPO）和SFT6组（SCF＋FL＋TP0＋IL-6）。结果表明，和对照组相比，SF组合仅能低水平支持脐血造血细胞扩增，加入TPO后即SCF／FL／TPO组合能有效的扩增脐血细胞，但SFT和SFT6两组之间差异却无明显发生（P〉0．05）；SF，SFT和SFT63组的细胞因子组合均可提高脐血CD34^＋细胞CD49d，CXCR4的表达，但3组之间差异无显著性（P〉0．05）。结论：SF组合可协同扩增人造血细胞，但协同作用较弱；TPO在脐血造血干／祖细胞体外扩增中起重要调节作用，而IL-6作用不显著；SCF／FL／TPO 3种因子组合不仅可促进脐血造血祖细胞的扩增，而且可上调脐血造血细胞CD49d，CXCR4表达。     

供者特征对脐带血造血功能的影响

目的 分析可能影响脐带血造血功能的供者特征.方法 对广州脐带血库1998年6月至2008年12月保存的4 358份脐带血的供者特征(包括母亲年龄、分娩方式、妊娠期、婴儿体重、婴儿性别)和脐带血采集量及造血功能的指标(包括总有核细胞数、CD34+细胞、干细胞集落等)进行相关性分析.结果 婴儿体重、分娩方式及婴儿性别是影响脐带血采集量、总有核细胞数、CD34+细胞数、CFUs及CFU-GM的主要因素.随着婴儿体重的增加,脐带血采集量、总有核细胞数、CD34+细胞数、CFUs、CFU-GM均呈上升趋势(P=0.000).阴道分娩时脐带血的采集量虽然低于刮宫产(P=0.000),但总有核细胞数、CD34+细胞数、CFUs,CFU-GM均高于剖宫产(P=0.000).女婴脐带血中总有核细胞数含量高于男婴(P=0.000),但脐带血采集量(P=0.000)、CD34+细胞数(P=0.002)均低于男婴.随着妊娠期的延长,脐带血中总有核细胞数增加(P=0.000),但CD34+细胞数减少(P=0.001).结论 某些脐带血供者特征对脐带血造血功能指标有积极影响.     

Viability does not necessarily reflect the hematopoietic progenitor cell potency of a cord blood unit: results of an interlaboratory exercise

BACKGROUND: Clinical transplant outcome with umbilical cord blood (UCB) as source of hematopoietic progenitor cells (HPCs) is, among other factors, determined by the total number of viable nucleated cells and/or CD34+ cells in the unit. Quantitative and qualitative losses by processing and cryopreservation and by thawing and washing before transfusion may occur, however. Another reason for a discrepancy between the number of cells in the unit released by the cord blood bank and found in the transplant center may be technical differences in cell counting methods between the two sites. STUDY DESIGN AND METHODS: With the collaborative group for Biomedical Excellence for Safer Transfusion (BEST), an interlaboratory exercise was conducted among nine sites for thawed UCB variables: total nucleated cells, CD34+ cells, viability, and HPC cultures. Three frozen UCB samples were shipped, with instructions for thawing, counting, and HPC plating. RESULTS: Unexpectedly samples arrived at all nine receiving centers without detectable hematopoietic progenitor colony-forming cells. Nevertheless, wide interlaboratory ranges for viability were obtained. The proportion of viable cells was found higher with manual methods, but all viability assays used in the study overestimated functional progenitor cells. CONCLUSIONS: The results underscore the complexity of evaluation of frozen-thawed cord blood cells and the need for standardization of assessment.     

人脐血及骨髓淋巴细胞免疫表型的比较及其意义

为比较脐血和骨髓淋巴细胞及祖细胞分化抗原，通过流式细胞术（FCM）双标法对38份脐血及10份骨髓免疫细胞表型进行了分析研究。研究发现：（1）脐血及骨髓淋巴细胞中均测到稚淋巴细胞（CD3^-CD4^ )，且前中含量较多，但脐血细胞毒T细胞含量（CTL，CD3^ CD16^ 56^ )低于骨髓；（2）脐血中NK细胞（CD3^-CD16^ 56^ )比例高于骨髓；（3）脐血有核细胞中CD34^ 细胞的比值接近于骨髓，但脐血CD34^ 细胞中髓系祖细胞（CD34^ CD13^ ,CD34^ HLA-DR^ )及淋巴系祖细胞（CD34^ CD19^ )含量均低于骨髓，结论：（1）脐血免疫细胞具有不成熟性，这估计是脐血移植后GVHD程度轻的主要原因；（2）脐血淋巴细胞中NK细胞含量较高，推测脐血移植后移植物抗白血病效应（GVL）并不会降低；（3）脐血CD34^ 细胞中髓系祖细胞及淋巴系祖细胞比例均低于骨髓，可能是脐血移植后造血及免疫重建速度较慢的原因之一。     

人脐血CD34+CD38-细胞免疫表型和染色体核型特征分析

目的 分离脐血干／祖细胞(CD34^ CD38)进行体外长期培养，观察分析其增殖、细胞表面分子标志和染色体核型的特征。方法 用流式细胞仪分选CD34-FITC和CD38-PE标记的CD34^ CD38脐血原始细胞，在含细胞生长因子IL-3、IL-6、GM-CSF、EPO、SCF和胰岛素样生长因子的干细胞培养基中培养6个月，用流式细胞术检测体外培养30d的干／祖细胞表面标记，并用G显带方法分析其染色体核型。结果 在一定培养条件下，经7～12d培养，脐血干／祖细胞(CD34^ CD38)开始增殖。培养6个月后，每孔接种1个细胞，细胞数增殖至250～350个；每孔接种10个细胞，细胞数可增殖至400～500个。每孔接种1个细胞其细胞增殖峰持续时间(8～9代)比接种10个细胞(6～7代)长：经体外长期培养增殖，细胞仍强烈显示十／祖细胞表面分子标记(CD34^ CD38^-)；细胞染色体数目、结构未见异常。结论 脐血干／祖细胞(CD34^ CD38^ )经体外特异性培养增殖，可为大量脐血干／祖细胞移植提供细胞来源。