免疫磁珠法分选骨髓CD34^＋细胞：纯度影响因素的分析

背景：免疫磁珠分选技术已成为分选CD34^＋细胞的主要方法之一,在长期实验中发现诸多因素影响其分离纯度,包括磁珠与细胞孵育时干冰与碎冰的使用、磁珠与细胞孵育及分离时摇床的摇摆方向、红细胞裂解液裂解单个核细胞的使用等。目的：在免疫磁珠分选CD34^＋细胞过程中分别对上述影响因素进行分析改进,以提高分选CD34^＋细胞的纯度。设计、时间及地点：细胞学体外对照观察,于2008-07/2009-03在太原市中心医院皮肤科实验室完成。材料：骨髓象筛选正常的人骨髓29份,由太原市中心医院血液科提供,取材均经患者同意。CD34^＋免疫磁珠为Dynal公司产品。方法：采用淋巴细胞分离液密度梯度离心法分离培养人骨髓单个核细胞,用免疫磁珠法分选骨髓CD34^＋细胞,对其分选过程中的主要环节如冰块与碎冰、摇床的种类以及红细胞裂解液的使用进行方法改进。方法1：按试剂盒提供方法进行磁珠常规分选CD34^＋细胞。方法2：在方法1基础上,用碎冰替代冰块进行磁珠与单个核细胞孵育。方法3：在方法2基础上,用万向摇床替代水平摇床。方法4：在方法3基础上,向单个核细胞中预先加入红细胞裂解液裂解残留红细胞。主要观察指标：通过流式细胞仪检测不同方法分选出的CD34^＋细胞纯度。结果：随着不同环节的逐渐改进,CD34^＋细胞纯度由（32.7±6.6）%升至（84.5±5.2）%,方法1,2,3,4所分选出的CD34^＋细胞纯度逐渐升高,组间方差分析差异显著（F=76.209,P〈0.01）,Bonferroni法两两比较差异亦有非常显著性意义（P〈0.01）。结论：碎冰、万向摇床及红细胞裂解液的使用能有效提高免疫磁珠法分选人骨髓CD34^＋细胞的纯度。     

CD34+细胞分选及临床应用

CD34+是造血干细胞的免疫表型。采用某种方法选择CD34+细胞,选择后CD34+细胞浓集,其百分率有较大增高,并可以有效去除T细胞及某些恶性肿瘤细胞。选择CD34+细胞移植可以减少异基因移植后急性、重度移植物抗宿主病(GVHD),并可能减少自体移植后原发病复发。本文介绍了造血干细胞的免疫表型,生物学特性及CD34+细胞分选方法、临床应用等方面的研究进展。     

免疫磁性分选系统大量分选CD34+细胞的实验研究

CD3 4 细胞的分离纯化在自体外周血干细胞、异基因骨髓移植 /外周血干细胞移植及干细胞研究中具有重要的应用价值。为了摸索大量分选CD3 4 细胞的方法 ,本研究应用Isolex3 0 0i磁性分选系统富集CD3 4 细胞 ,采用流式细胞术监测分选前后细胞表面标志 ,经CD3 4 细胞体外增殖实验及克隆形成实验验证分选获得的CD3 4 细胞生物学活性。结果显示 ,所完成的 5例次自体外周血富集CD3 4 细胞时 ,先收获单个核细胞约 ( 3 .5 -6.0 )× 10 10 ,CD3 4 细胞占单个核细胞百分率为 ( 0 .5 5 - 1.2 ) % ;收获的CD3 4 阳性细胞总数为 ( 2 - 3 )× 10 8,纯度为 ( 75 - 85 ) % ,回收率为 ( 4 0 - 65 ) %。体外实验表明 ,在SCF IL 3 FL TPO EPO存在下 ,经过 3 - 4天培养 ,可扩增 2 - 3倍 ;经CFU GM、BFU E集落形成实验显示具有形成集落的能力 ,证实分选后细胞具有造血祖细胞生物活性。结论 :应用Isolex3 0 0iCD3 4 细胞分选仪可以高效大量富集CD3 4 细胞 ,适于临床应用。     

影响外周血CD34+细胞纯化的相关因素

背景:造血干细胞具有良好的自我复制和更新的能力,CD34+细胞具备有造血干细胞的标志.目的:分析影响外周血CD34+细胞纯化的因素.方法:90例患者,经粒细胞集落刺激因子5 μg/(kg·d)动员1～3 d后,应用COBE血细胞分离机采集外周血单个核细胞液80～100 mL,经Clini MACS免疫磁珠分选技术纯化CD34+细胞.结果与结论:90例CD34+细胞平均数为(1.73±1.15)×107,经流式细胞仪分析,CD34+细胞阳性率大于80%.COBE血细胞分离机单次收集的循环血量在980～1 100 mL时,利于CD34+细胞收集(P=0.005);动员后白细胞浓度在(16～21)×109 L-1时,利于CD34+细胞收集(P < 0.05);中间细胞和淋巴细胞总比例超11%时,利于CD34+细胞收集(P < 0.05);单个核细胞液血小板小于2 100×109 L-1时,利于CD34+细胞的收集(P < 0.05);年龄小于16岁,CD34+细胞数高(P=0.003);CD34+细胞抗体的温度、磁性标记及细胞处理时离心力的大小,均有影响.结果提示,经Clini MACS免疫磁珠细胞分选技术纯化的CD34+细胞能满足临床需要,实验稳定性好,重复性好;注重相关因素的影响,可提高纯化的CD34+细胞数量.     

致敏小鼠CD4~+ CD25~+调节性T细胞磁珠分选及体外扩增

本研究探讨致敏小鼠CD4+ CD25+调节性T细胞的分选及体外扩增。流式细胞术检测致敏小鼠及正常小鼠体内CD4+ CD25+ Treg细胞水平,免疫磁珠分选方法从小鼠脾细胞中分选出CD4+T细胞、CD4+ CD25+ Treg细胞和CD4+ CD25-T细胞,负载抗CD3/CD28单克隆抗体MACSiBead联合IL-2共同刺激CD4+ CD25+ Treg细胞进行体外扩增培养,用0.4%台盼蓝染色并计数检测细胞的活性,流式细胞术检测分选后细胞纯度、主要表面标记及Foxp3基因的表达。结果表明:致敏小鼠体内CD4+ CD25+ Treg水平较正常小鼠升高(P<0.05)。分选出CD4+ CD25+ Treg细胞纯度平均达到87%,细胞活性大于97%,高表达Foxp3基因。体外扩增2周后细胞数扩增倍数能够达到42倍,CD4+ CD25+ Treg细胞所占比例为85.32%,Foxp3表达由(76.92±1.72)%稍下降至(75.33±2.11)%(P>0.05)。结论:免疫磁珠分选法能够分选出高纯度的CD4+ CD25+ Treg细胞,该分选方法不影响分选靶细胞的细胞活力;体外成功扩增了CD4+ CD25+ Treg细胞,扩增后的CD4+ CD25+ Treg细胞表面标记及Foxp3基因表达无明显改变。     

影响骨髓血CD34+细胞纯化数量的因素分析

目的 探讨影响骨髓血CD34+细胞纯化的因素.方法 45例进行性肌营养不良患者的骨髓血标本,在去或不去RBC和PLT的状况下,经Clini MACS免疫磁珠分选技术纯化CD34+细胞并计数其数量.结果 RBC和PLT的多少对CD34+细胞的纯化有显著的影响,差异具有统计学意义.结论 在进行Clini MACS免疫磁珠分选技术对骨髓血CD34+细胞纯化时,去除RBC及其碎片和PLT,可以提高CD34+细胞纯化的数量.     

免疫磁珠分离及流式细胞仪分选纯化外周血CD34+/CD90+干细胞

目的　建立人外周血CD34 细胞及其亚群的纯化分离方法。方法　用干细胞采集仪收集了 3例病人的外周血干细胞 ,应用免疫磁珠分离柱快速分离其中的CD34 细胞 ,随后采用分选型EPICSElite流式细胞仪进一步分选出CD34 /CD90 双阳性早期造血干细胞。结果　免疫磁珠分离纯化后CD34 干细胞的纯度可达 83%～ 95 % ,回收率 5 4 %～ 71% ,活细胞率 >95 %。流式细胞仪分选后的CD34 /CD90 细胞纯度可达 90 %以上 ,回收率在 4 0 %～ 5 0 % ,生存率 >95 %。起始标本中CD34 细胞含量越高 ,纯化所得到的干细胞纯度和回收率越高。两种纯化后的干细胞在形态学上有明显的不同。结论　联合应用免疫磁珠分离和流式细胞仪分选 ,可以较高的回收率快速纯化高纯度的CD34 细胞及其亚群     

影响外周血CD34^＋细胞纯化的相关因素

背景：造血干细胞具有良好的自我复制和更新的能力,CD34^＋细胞具备有造血干细胞的标志。目的：分析影响外周血CD34^＋细胞纯化的因素。方法：90例患者,经粒细胞集落刺激因子5μg/（kg·d）动员1～3d后,应用COBE血细胞分离机采集外周血单个核细胞液80～100mL,经CliniMACS免疫磁珠分选技术纯化CD34^＋细胞。结果与结论：90例CD34^＋细胞平均数为（1.73±1.15）×107,经流式细胞仪分析,CD34^＋细胞阳性率大于80%。COBE血细胞分离机单次收集的循环血量在980～1100mL时,利于CD34^＋细胞收集（P=0.005）;动员后白细胞浓度在（16～21）×109L-1时,利于CD34^＋细胞收集（P〈0.05）;中间细胞和淋巴细胞总比例超11%时,利于CD34^＋细胞收集（P〈0.05）;单个核细胞液血小板小于2100×10^9L^-1时,利于CD34^＋细胞的收集（P〈0.05）;年龄小于16岁,CD34^＋细胞数高（P=0.003）;CD34^＋细胞抗体的温度、磁性标记及细胞处理时离心力的大小,均有影响。结果提示,经CliniMACS免疫磁珠细胞分选技术纯化的CD34^＋细胞能满足临床需要,实验稳定性好,重复性好;注重相关因素的影响,可提高纯化的CD34^＋细胞数量。     

阵发性睡眠性血红蛋白尿症患者骨髓CD34+CD59+细胞与CD34+CD59-细胞生物学性能的研究

目的 探讨阵发性睡眠性血红蛋白尿症(PNH)患CD34^ CD59^ 细胞的特性及PNH克隆呈优势造血的可能原因，以探索PNH发病的内在机制。方法 用免疫磁珠吸附法富集纯化CD34^ 细胞，再用流式细胞仪分选出PNH患的CD34^ CD59^ 细胞、CD34^ CD59^ 细胞及正常对照CD34^ 细胞。分别进行体外扩增液体培养2周，并对扩增前、后的细胞进行半固体培养。结果 ①PNH患CD34^ CD59^ 细胞与正常对照CD34^ 细胞形成集落形成单位(CFU)均在第7天达到扩增高峰，并且扩增后的细胞仍能保持CD59抗原，无GPI锚连蛋白的丢失。②正常对照的CD34^ 细胞在生存、增殖、形成CFU及扩增能力上均明显强于FHN患的CD34^ CD59^ 细胞及CD34^ CD59^-细胞.③PNH患CD34^ CD59^ 细胞及CD34^ CD59^ 细胞体外半固体培养，其形成CFU的能力无明显差异。④PNH患CD34^ CD59^ 细胞及CD34^ CD59^ 细胞在SCF IL3 IL6 FL Tpo及SCF IL3 IL6 FL Tpo Epo组合下液体培养，其生存、增殖、扩增等能力上均无明显差异。但在SCF IL3 IL6 FL Tpo Epo GM-CSF组合下液体培养，CD34^ CD59^ 细胞的生存、增殖、扩增能力均明显强于CD34^ CD59^ 细胞。结论 ①正常对照的CD34^ 细胞在生存、增殖、形成CFU及扩增能力上均明显强于PNH患的CD34^ CD59^ 细胞。②PNH患CD34^ CD59^ 细胞及CD34^ CD59^ 细胞体外半固体培养，以及在SCF IL3 IL6 LF Tpo及SCF IL3 IL6 FL Tpo Ep组合下液体培养，其生存、增殖、扩增等能力上均无明显差异，说明CD34^ CD59^ 细胞在造血能力上并无内在的优势。GM—CSF或许是使PNH克隆呈造血优势的原因之一。     

Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield

BACKGROUND: In immunomagnetic selection of CD34+ cells from HPC transplants, not all factors that affect yield and purity of CD34+ cells are known. METHODS: Forty-three consecutive procedures of immunomagnetic selection of CD34+ cells from peripheral blood HPCs and bone marrow harvests (autologous harvests, n = 27; allogeneic harvests; n=16) were performed by use of a cell selection system (Isolex 300i, Baxter Immunotherapy). The composition of the starting component and the subsets of CD34+ cells were analyzed for correlation with the yield and purity of the final component. RESULTS: The mean purity of the final components was 84.3 percent (range, 27-99%), and the mean yield was 51.4 percent (range, 9.4-80. 4%). Partial regression analysis showed that, among the factors correlating with purity and/or yield, the RBC volume in the starting fraction had the highest predictive impact on the purity and yield of CD34+ cells, even after the exclusion of procedures using bone marrow harvests as an HPC source (beta coefficient, -0.704; p = 0. 001). CONCLUSION: The use of the Isolex 300i system allows efficient recovery of CD34+ cells in routine selection procedures. The volume of RBCs in the starting component should be minimized to ensure a high yield and purity of the final component.     

冠状动脉自体骨髓CD34＋干细胞移植治疗心绞痛：随机对照临床分析

背景:大量基础和临床研究结果提示,骨髓CD34+干细胞具有明显的促进血管新生作用.经作者查新检索,目前国际上仅有1篇小样本心腔内沣射骨髓CD34+下细胞治疗心绞痛的报道.目的:评价冠状动脉内CD34+干细胞移植治疗心绞痛的安全性和有效性.设计:回顾性病例分析.对象:选取2007-07/2008-07解放军北京军区总医院心内科住院的32例心绞痛患者,按患者意愿分为治疗组和对照组,移植前1个月内均无急性心肌梗死病史,心绞痛加拿大心血管学会分级为Ⅱ～Ⅳ级,两组患者基线资料比较无统计学差异(P＞0.05).方法:治疗组患者在接受最佳药物治疗的基础上,于常规PCI术后,行冠状动脉内自体骨髓CD34+干细胞移植,于冠状动脉病变近端注入CD34+干细胞悬液15 mL,细胞数为(1.0～6.1)×106.对照组患者仅接受最佳药物治疗和导管置入.主要观察指标:移植后两组患者的疗效指标比较及心肌血液灌注情况.结果:骨髓干细胞移植后,治疗组心绞痛发作频率、硝酸甘油的用量、运动时间及加拿大心血管学会心绞痛分级等各项疗效指标均明显优于对照组(P＜0.001～0.05),且移植后6个月时的治疗效果优于移植后3个月.移植后6个月,治疗组患者心肌灌注缺损面积明显小于对照组(P＜0.01).随访6个月内,未观察到因冠状脉动脉内注射而导致的心肌梗死、心肌酶升高、心肌穿孔、心包积液、室性心动过速或室颤等严重不良事件发生.结论:冠状动脉内注射自体骨髓CD34+干细胞后,可显著改善心绞痛患者心肌血液灌注,安全有效.     

Age and stress related phenotypical changes in bone marrow CD34+ cells

Objective. Phenotypical changes in the human bone marrow (BM) due to age and stress have not so far been properly addressed in the literature. In the present study, we compared CD34⁺ BM cells between older and young volunteers. The influence of stress on CD34⁺ cell phenotype in older patients was investigated in an age‐matched group with acute myocardial infarction (AMI). Cytokines thought to influence BM CD34⁺ cell homeostasis were also analysed. Material and methods. BM mononuclear cells of 10 older volunteers and of 7 young volunteers (18–25 years), as well as 22 AMI patients, were analysed by flow cytometry for the following markers: CD34, CD38, CD117 (c‐kit) and CD133. Blood samples were analysed for CRP, IL‐6, MCP‐1, IL‐8, MMP‐9, TIMP‐1 and TNFα by ELISA methods. Results. Significantly higher numbers of CD34⁺ CD38^? cells (both absolute and relative) were observed in older volunteers than in young volunteers and AMI patients. Higher numbers of immature progenitors, namely CD34⁺CD38^? cells and CD34⁺CD38^?CD117⁺CD133⁺ cells, were observed among older volunteers compared to the other groups. However, the relative number of CD34⁺ cells lacking CD38 expression or expressing CD133 was higher in the old volunteers and AMI patients. None of the circulating factors investigated correlated with any of the cell population yields. Conclusion. In this study, we found that the absolute and relative numbers of BM CD34⁺CD38^? progenitor cells increase with age. The increment is attenuated in patients with AMI.     

阵发性睡眠性血红蛋白尿症患者骨髓CD34+CD59+细胞的体外扩增

目的研究阵发性睡眠性血红蛋白尿症(PNH)患者CD34+CD59+细胞的分离、纯化及其体外扩增的条件和性能,为探索PNH新的治疗途径提供实验依据.方法利用免疫磁珠-流式细胞仪二步分选法,从PNH患者骨髓中分选出CD34+CD59+细胞,然后对CD34+CD59+细胞在不同造血生长因子组合条件下,进行体外扩增培养2周.结果体外扩增的最适宜的生长因子组合为SCF+IL-3+IL-6+FL+Tpo+Epo,最适宜的扩增时机为第7天,在此条件下,CD34+CD59+细胞的扩增倍数为22.42±3.73倍.CD34+CD59+细胞在扩增以后,仍保持较好的形成集落形成单位的能力,但是其向多系分化的潜能有所下降.结论 PNH患者CD34+CD59+细胞能够进行体外扩增.按照最佳扩增条件,在对PNH患者进行自体骨髓移植或自体外周血干细胞移植时有一定的应用价值.     

Correlation of CD34+ cell counts with volume of bone marrow collected for allogeneic bone marrow harvests

The quantity of bone marrow collected for allogeneic bone marrow transplantation is based on collecting 10 to 15 cc of bone marrow/kg of recipient weight. We hypothesized that the percentage of CD34+ cells collected during a bone marrow harvest decreased at the end of the harvest because of increasing amounts of peripheral blood contamination. We performed a prospective, blinded study in which we measured CD34+ percentages and cell counts at 200-cc intervals during bone marrow harvests from 11 consecutive human leukocyte antigen (HLA)-matched sibling bone marrow donors. We observed that the percentage of CD34+ cells in aspirated bone marrow did not vary significantly from the start to the end of the bone marrow harvest, and the total number of CD34+ cells/kg increased in a linear fashion, thus disproving our original hypothesis. In conclusion, the percentage of CD34+ cells in aspirated bone marrow will remain constant throughout a bone marrow harvest.     

T lymphocyte differentiation in vitro from adult human prethymic CD34+ bone marrow cells

Pluripotent lymphohematopoietic stem cells are probably confined to bone marrow cells expressing CD34 surface molecules. To investigate the capacity of adult human CD34+ bone marrow cells to differentiate along the T lymphoid lineage, we plated purified CD34+ cells from healthy adults in liquid culture on adherent thymic stromal cells prepared from HLA- or blood group-mismatched postnatal thymic tissue. We show that purified CD34+CD3-CD4-CD8- bone marrow cells contained progenitors with the ability to differentiate into CD4+ and CD8+ T lymphocytes expressing surface (s)CD3 and T cell receptor alpha/beta in vitro. These progenitors were found in the CD34+CD2+sCD3-CD4-CD8-, CD34+CD7+sCD3-CD4-CD8-, and CD34+CD2+CD7+sCD3-CD4-CD8-, as well as in the CD34+CD2-sCD3-CD4-CD8-, CD34+CD7-sCD3-CD4-CD8-, and CD34+CD2-CD7- sCD3-CD4-CD8- subsets, indicating that T lymphocyte progenitors sensitive to signals mediated by thymic stroma in vitro are not restricted to CD34+ cells already coexpressing early T lymphocyte- associated markers. Finally, we show that T lymphopoiesis was enhanced by c-kit ligand.     

人正常骨髓CD34＋造因细胞的形态学与细胞化学特征

OBJECTIVE: To explore the cytomorphological and cytochemitry features of CD34+ hematopoietic cells. METHODS: Highly purified CD34+ hematopoietic cells isolated from human bone marrow by a two-step method of immunomagnetic beads-FACS sorting were comprehensively examined. RESULTS AND CONCLUSION: CD34+ hematopoietic cells could be sorted into three types in light of morphology and cytochemical staining. Type I was considered as candidates for stem cells,which were most blastlike in morphology and slightly larger than lymphocytes in size with negative reactions to all cytochemical stainings. Type II was considered as multipotent progenitor cells, with sizes similar to small lymphocytes and negative reactions to all cytochemical stainings. Type III was identified as committed progenitor cells whose sizes were heterogeneous and whose reactions to cytochemical stainings ranged from +/- to + +.