粒细胞集落刺激因子在自体外周血干细胞移植治疗缺血性下肢血管病中的作用

目的：观察缺血性下肢血管病患者进行自体外周血干细胞移植时。应用粒细胞集落刺激因子后细胞成分的变化以及对自身身体状况的近期影响。 方法：选取2004-11／2005—04解放军第四六三医院内分泌科收治的126例接受粒细胞集落刺激因子动员的缺血性下肢血管病患者，全部接受皮下注射粒细胞集落刺激因子5-12μg（kg&;#183;d），连续4-5d。为防止血黏度增加引起心脑血管意外，在干细胞动员的同时应用低分子肝素钙5000u，皮下注射1次／d。连续4～5d。每天监测血细胞计数和凝血3项。同时采用流式细胞仪监测外周血中CD34^＋细胞数。观察并记录动员后及采集过程中、后出现的毒副反应。 结果：按意向处理分析。实验纳入126例缺血性下肢血管病患者。全部进入结果分析。①全部患者动员过程中外周血象的变化：粒细胞集落刺激因子动员前白细胞数量为（5．35&;#177;1．64）&;#215;10^9L^-1，动员第5天为（42．17&;#177;18．56）&;#215;10^9L^-1．第6天为（44．23&;#177;17．47）&;#215;10^9L^-1。动员后比动员前提高5～13倍（P〈0．01）；血红蛋白和血小板动员前后无明显变化。（爹全部患者动员后采集细胞悬液的情况：37例患者于动员第5天进行采集，单个核细胞数值和CD34^＋百分数分别为（432．68&;#177;89．36）&;#215;10^9L^-1和（0．87&;#177;0．38）％；其余89例均于第6天进行采集，单个核细胞数值和CD34^＋百分数分别为（463．71&;#177;58．33）&;#215;10^9L^-1和（0．90&;#177;0.35）％，两者基本相近（P〉0．05）。③性别、年龄和体质量对单个核细胞数值和CD34^＋细胞百分数的影响：男性患者采集的单个核细胞数值高于女性患者（P〈0．05）．而单位体质量的CD34^＋细胞数值男女基本相近；以年龄55岁为界，大于55岁和小于55岁的患者差异显著（P〈0．05）；高体质量患者采集的单个核细胞数值高于低体质量患者（P〈0．05），而单位体质量的CD34^＋细胞数值基本相似。④不良事件和副反应：主要的不良反应有骨痛、周身肌肉酸痛、乏力、头痛、失眠、食欲下降、恶心呕吐、低热。采集过程中可能出现口周、面部或四肢麻木，一般停药2～4d症状即可消失。 结论：缺血性下肢血管病患者进行自体外周血干细胞移植时。粒细胞集落刺激因子作为有效动员剂，可有效动员单个核细胞和CD34^＋细胞。绝大部分患者能够耐受．但廊任用一定剂量的抗凝剂预防不良反席的发毕．     

异基因造血干细胞移植CD34^＋CD61^＋巨核系前体细胞与血小板植入的相关分析

本研究通过定量测定G—CSF动员后外周血及骨髓采集物的CD34^＋CD61^＋细胞，以探索巨核前体细胞CD34^＋CD61^＋亚群输注量与异基因外周血和／或骨髓移植后血小板恢复时间的相关性。24例不同血液病患者接受HLA全相合同胞、非血缘供者或单倍体相合同胞造血干细胞移植。20例可评估的患者依移植方法不同分为HLA全相合组和单倍体相合组，HLA全相合组采用PBSC移植方案，单倍体相合组采用PBSC＋BM联合移植方案，对外周血干细胞和骨髓样本CD34^＋CD61^＋亚群通过流式细胞仪立即测定或隔夜保存后测定。结果表明，单倍体相合组输注CD34^＋、CD34^＋CD61^、CD34^-CD61^＋细胞数中位值分别为6．24×10^6／kg（1．53—20．48）、66．19×10^4／k（8．16—493．83）、34．38×10^6／kg（14．71—109．16）；HLA全相合组中位值分别为4．88×10^6／kg（1．00—8．24）、14．16×10^4／kg（11．63—96．87）和13．50×10^6／kg（1．74—35．61）。中性粒细胞计数（ANC）〉0．5×10^9／L和血小板计数〉20×10^9／L的中位时间，单倍体相合组分别为18．5（11．00—29．00）和16．5（9．00—35．00）天；HLA全相合组为14．5（9．00—24．00）和10．5（6．00—37．00）天，血小板的植入时间在两组差别无显著性，中性粒细胞植入时间在HLA全相合组和单倍体相合组差异有显著性（p=0．048），对于两组中CD34^＋细胞量〉2×10^6／kg的受者血小板的植入时间分析，两组差别有显著性（p=0．006）。CD34^＋CD61^＋亚群与血小板植入的相关性明显优越于CD34^＋细胞，可评估20例（r=-0．449p=0．047CD34^＋细胞群），单倍体相合组12例CD34^＋CD61^＋亚群（r=-0．768p=0．004），HLA相同纽8例CD34^＋CD61^＋亚群（r=-0．747p=0．033），CD34^＋CD61^＋亚群与血小板的植入时间呈负相关关系，输注CD34^＋CD61^＋亚群细胞量大，血小板的植入时间缩短。结论     

不同来源人造血干/祖细胞在NOD/SCID小鼠体内归巢能力的差异

目的 比较不同来源的人造血干／祖细胞在NOD／SCID小鼠体内归巢能力的差异性，并探讨其体内归巢能力与膜表面归巢相关分子CXCR4表达水平的相关性。方法 采用流式细胞术（FACS）检测新鲜脐血、冻存脐血、动员后外周血（mPB）及骨髓来源的CD34^＋细胞表面CXCR4表达水平；将荧光染料CFSE标记的人CD34^＋细胞移植人接受照射的NOD／SCID小鼠，移植后20小时检测已归巢于NOD／SCID小鼠骨髓及脾脏中不同来源的人CD34^＋细胞，计算其相应的骨髓及脾脏归巢效率；并将小鼠股骨制成组织切片，荧光显微镜下观察人CD34^＋细胞在小鼠骨髓腔内的分布。结果 新鲜脐血、冻存脐血、mPB和骨髓CD34^＋细胞膜表面CXCR4表达阳性率分别为（49.52±1．12）％。（46．12±2．29）％，（48．50±2．48）％和（65．39±1．27）％，CD34^＋细胞在NOD／SCID小鼠骨髓的归巢效率分别为（3．00±0．44）％，（2．84±0．46）％，（4．06±0．70）％及（5．76±0．52）％；在脾脏的归巢率分别为（1．88±0．12）％，（1．80±0．15）％，（1．90±0．22）％，（2．16±0．34）％。归巢的CD34^＋细胞主要分布于小鼠股骨的骨内膜区域。结论 脐血CD34^＋细胞膜表面CXCR4水平低于mPB和骨髓。经冻存复苏后脐血CD34^＋细胞膜表面CXCR4水平略有下调。脐血CD34^＋细胞在照射NOD／SCID小鼠的骨髓归巢效率低于mPB和骨髓。     

骨髓干细胞动员疗法对心肌梗死大鼠心脏的促血管生成作用

目的探讨干细胞动员疗法对心肌梗死大鼠冠脉新生血管形成的影响及其作用机制。方法结扎Wistar大鼠左冠状动脉制作急性心肌梗死模型，应用粒细胞集落刺激因子动员自体骨髓干细胞释放并归巢于缺血心肌，建模后第24h、48h和4周时处死大鼠，取出心脏，HE染色检测梗死面积，免疫组化方法观察心肌梗死灶、边缘区和正常心肌组织CD34^＋胞、血管内皮生长因子（VEGF）及其受体（Flk—1）的表达以及Ⅷ因子表达。结果使用干细胞动员剂后，动员组大鼠心肌梗死区可见CD34^＋浸润，心肌梗死面积明显减小，梗死灶及周围组织中微血管密度、VEGF及其受体的表达量均明显高于AMI组及假手术对照组。结论骨髓干细胞动员疗法在大鼠AMI模型中，通过动员骨髓干细胞归巢于梗死灶内，有效促进微血管形成；通过上调VEGF和VEGF受体Flk-1的表达，促进血管再生，促进缺血心脏功能恢复。     

急性心肌梗死后CD34^＋干细胞自体动员的意义

目的：已有理论提出急性心肌梗死后骨髓和外周血中的CD34^＋干细胞具有自身动员的潜能，观察这一潜能的变化特征及其对心肌梗死组织再生能力的影响。方法：实验于2004-09／2005-02在阜外心血管病医院完成。①实验动物：雄性SD大鼠40只。随机数字表法分为心肌梗死组、假手术组，20只/组。②实验方法：心肌梗死组大鼠采用冠状动脉结扎法建立心肌梗死模型。心电图ST段抬高或有室性心律出现，前壁心肌呈苍白色为造模成功。假手术组仅作开胸手术，前降支不予结扎。③实验评估：于心肌梗死后3,7，14，28d，流式细胞仪检测骨髓和外周血中CD34^＋干细胞的含量。用免疫组化方法检测梗死心肌组织中的Ki67细胞和毛细血管数量。结果：①外周血及骨髓CD34^＋干细胞含量的变化：心肌梗死组外周血中的CD34^＋干细胞数量于造模后3d开始上升，7d后明显高于假手术组（P〈0.01），至14,28d时逐渐回落至假手术组水平（P〉0.05）。心肌梗死组骨髓中的CD34^＋干细胞数量于造模后各时间点始终无明显变化（P〉0.05）。②组织学评定：心肌梗死组梗死区Ki67细胞和毛细血管数量于造模后3d开始增多，7d时明显多于非梗死区（P〈0.05）；至14，28d梗死区Ki67细胞数量明显少于造模后7d（P〈0.05），毛细血管数量的减少不明显（P〉0.05）。免疫组化染色显示少数Ki67细胞分化为血管内皮细胞，未见向心肌细胞分化。③相关性分析：梗死区Ki67细胞、毛细血管数量于造模后7d与外周血中CD34^＋干细胞数量呈显著正相关（r=0.913，P=0.021；r=0.887，P=0.035）。结论：机体CD34^＋干细胞的自体动员、增殖反应的潜能随急性心肌梗死时间的延长而逐渐减弱，自体动员的干细胞功能尚不足以达到修复梗死心肌组织的效果。     

rhG-CSF动员健康供者CD34^＋细胞产率的影响因素分析

为探讨rhG—CSF动员健康供者年龄、性别等因素对采集物CD34^＋细胞产率的影响,分析61例健康供者外周血采集物CD34^＋细胞数量与自身特点的相关性,并进行多因素回归分析。以供者性别、年龄、身高、体重、体重指数（BMI）和采集时间作为研究参数,外周血采集物单核细胞计数、CD34^＋细胞占有核细胞百分比、CD34^＋细胞计数、CD34^＋细胞每公斤体重（供者）计数的均值作为研究变量。结果表明,供者年龄是影响CD34^＋细胞产率主要因素,呈中等负相关（-0．60〈r〈-0．45,P〈0．005）。偏相关分析排除身高、体重、BMI的影响,年龄仍和CD34^＋细胞产率呈中等负相关（-0．50〈r〈-0．35,P〈0．02）。BMI仅与CD34^＋细胞每公斤体重计数呈微弱负相关（r=-0．297,P〈0．05）,性别对CD34^＋细胞产率无明显影响,CD34^＋细胞计数的差别仅出现在男性和女性低龄组（年龄〈35岁）间,男性身高、体重、BMI为CD34^＋细胞计数增加的有利因素。供者采集时间为给药后第4天采集,70％供者CD34^＋细胞产率达峰值。结论：年龄应作为供者选择的首要因素,性别、身高、体重和BMI对CD34^＋细胞产率的影响是次要的。     

体外不去T细胞单倍体造血干细胞移植后NK细胞免疫球蛋白样受体重建的影响因素

目的探讨造血干细胞移植（HSCT）后早期NK细胞免疫球蛋白样受体（KIR）恢复的影响因素。方法借助三色和四色荧光标记技术，用流式细胞术对24例行体外不去T细胞的HLA不相合HSCT的患者（移植前、＋30天、＋60天）及其供者外周血中NK细胞KIR的表达进行了测定，包括CD158a（KIR2DL1）、CD158b（KIR2DL2）和CD158e（KIR3DL1）；同时用流式细胞术对移植物中CD3、CD4、CD8、CD14、CD34的含量进行了测定。结果发生Ⅱ～Ⅳ度急性移植物抗宿主病（aGVHD）患者在＋30天NK细胞KIR的表达明显低于0～I度aGVHD患者，CD158b[分别为（19．27±9．40）％和（28．92±10．59）％，P=0．018]和CD158aCD158b[分别为（7．30±4．73）％和（14．26±9．71）％，P=0．016]尤为显著。多因素分析表明移植物中CD4^＋T细胞是引起aGVHD的危险因素。进一步的相关分析表明每千克体重输入的CD4^＋T细胞数与移植后早期NK细胞上CD158a、CD158aCD158b和CD158e的表达呈明显的负相关。结论体外不去T细胞的HLA不相合的HSCT中，不仅移植后aGVHD的发生或其相应的治疗措施抑制了移植后早期NK细胞上KIR的恢复；而且移植物中T细胞也直接或间接影响了移植后早期NK细胞上KIR的重建，进而影响了NK细胞功能的恢复。     

间充质干细胞对脐血CD34^＋细胞扩增及其细胞特性变化的影响

本研究探讨脐带间充质干细胞（MSC）对CD34^＋细胞（HSPC）体外扩增的支持作用及对CD34^＋细胞表面标志、归巢黏附分子、集落形成能力等干细胞特征变化的影响。用免疫磁珠法从新鲜分离的脐血单个核细胞分离CD34^＋造血干祖细胞（HSPC）；用MSC饲养层（feeder）制备经^137Cs照射的间充质干细胞饲养细胞（MSC feeder cells）。将CD34^＋细胞接种在不同的培养体系中，实验分为3组：HSPC＋CK组为培养液中加入细胞因子组合（SCF、FL和TPO），HSPC＋MSC组为CD34^＋细胞接种在MSC feeder上，HSPC＋MSC＋CK组同时加入细胞因子组合及MSC饲养细胞。培养后4、7、10、14天计数有核细胞总数（MNC），计算细胞扩增情况；用流式细胞术检测不同处理组间CD34^＋细胞及亚群免疫表型、归巢黏附分子和集落形成能力。结果表明：在2周的培养时间里，3组MNC和CD34^＋细胞均明显增加，MNC扩增数依次HSPC＋MSC＋CK组〉HSPC＋CK组〉HSPC＋MSC组。体外扩增10天内HSPC＋MSC＋CK组MNC得到大量的扩增，同时CD34^＋细胞的扩增亦较高。培养4天3组细胞CD34^＋比例较0天有明显下降（P〈0．01）；扩增后CD34^＋细胞比例：HSPC＋MSC组〉HSPC＋MSC＋CK组〉HSPC＋CK组（P〈0．01）；各组CD34^＋细胞亚型细胞比例有所不同，HSPC＋CK组4天时CD34^＋CD38^-细胞有一过性升高（62．71％），之后迅速降低，7天时为0．05％；HSPC＋MSC组7天时CD34^＋CD38^-细胞比例为18．92％，与HSPC＋CK组比较差异有统计学意义（P〈0．05）。从集落形成分析结果看出：MSC、细胞因子混合组扩增后细胞集落形成能力在不同时间点均维持在较高水平。结论：脐血CD34^＋细胞在体外短期培养（〈7天）下，MSC和细胞因子联合应用能同时使CD34^＋细胞得到明显的扩增并维持造血干祖细胞的生物学特征。     

CD4+CD25+调节性T细胞在特发性血小板减少性紫癜患者中的变化

目的探讨CD4^＋CD25^＋T细胞在特发性血小板减少性紫癜（ITP）患者发病机制中的作用。方法应用流式细胞术检测ITP患者外周血CD4^＋CD25^＋T细胞、CD4^＋CD25^highT细胞、CD4^＋FOXP3^＋T细胞、CD4^＋CD25^＋FOXP3^＋T细胞的数量；实时荧光定量PCR检测外周血FOXP3 mRNA的表达水平。将ITP患者和正常人CD4^＋CD25^highT细胞与自身CD4^＋CD25^-T细胞混合培养，检测CD4^＋CD25^high T细胞免疫抑制功能。结果ITP患者外周血中CD4^＋CD25^＋T细胞约占CD4^＋T细胞的（15．64±5．82）％，明显高于正常对照组（9．30±3．95）％（P〈0．01），CD4^＋CD25^high T细胞比例为（1．53±0．66）％，与对照组[（1．36±0．55）％]比较差异无统计学意义（P〉0．05）；CD4^＋FOXP3^＋T细胞和CD4^＋CD25^＋FOXP3^＋T细胞分别为（1．82±1．42）％和（1．25±0．94）％，均明显低于对照组[（3．90±1．37）％和（2．65±0．92）％]（P值均〈0．01）。ITP患者外周血FOXP3 mRNA表达水平较正常人明显下调（P〈0．01），CD4^＋CD25^high T细胞的抑制活性较正常人减弱（P〈0．01）。结论ITP患者中CD4^＋CD25^＋T细胞FOXP3表达水平降低，抑制活性减弱。     

骨髓腔内输注人造血干／祖细胞促进NOD-SCID小鼠体内造血功能的恢复

本研究比较经骨髓腔内输注（intra—bone marrow infusion,iBMI）及静脉输注（intravenous infusion,iVI）人脐血（cord blood,CB）造血千／祖细胞（HS／PC）对NOD～SCID受鼠体内造血重建的影响。将纯化的CB CD34^＋细胞移植到受亚致死剂量照射的NOD—SCID受鼠体内。受鼠随机分为3组：①iBMI组：骨髓腔内输注CD34^＋细胞5×10^5／只;②iVl组：尾静脉输注CD34^＋细胞5×10^5／只;③阴性对照组：输注PBS缓冲液。于异种移植后第3、5及第8周通过流式细胞术、聚合酶链式反应、免疫组织化学及造血祖细胞集落分析的方法比较人造血系统各系细胞植入率,通过二次移植实验比较NOD—SCID受鼠体内人HS／PC长期造血重建能力。结果表明：移植后第8周,iB—MI组受鼠骨髓、外周血及脾脏中人CD45^＋细胞比例均显著高于iVI组（P〈0．05）;iBMI组及iVI组移植的HS／PC均具有多向分化能力;移植后第8周,iBMI组受鼠骨髓中CD45^＋CD19^＋细胞、CD45^＋CD33^＋细胞、CD45^＋CD56^＋细胞及CD45^＋CD34^＋细胞比例均显著高于iVI组（P〈0．05）,CD45^＋CD14^＋细胞及CD45^＋CD41a^＋细胞比例亦高于iVI组（P〉0．05）。iVI组及iBMI组长期存活受鼠的肝脏、脾脏、肺脏、外周血、骨髓细胞中均可检测到人17号染色体α-卫星特异性片段。应用免疫组织化学法在移植后第8周的iBMI组受鼠的脾脏、肝脏和肺脏中均可检出人CD45抗原的表达.iBMI组受鼠的骨髓细胞各系集落总数于移植后第8周显著高于iVI组（P〈0．05）。二次移植后第6周,iVI组及iBMI组受鼠的各组织脏器中均可检出人17号染色体α-卫星特异性片段的表达。结论：与iVI相比,经iBMI移植人CBCD34^＋细胞至NOD—SCID受鼠可以促进HS／PC的造血重建及多向分化,提高植入率。     

自体外周血单个核细胞移植治疗下肢动脉缺血性疾病的研究

目的 分析59例下肢缺血性疾病患者进行自体外周血单个核细胞(MNC)移植的疗效及其与移植MNC数及CD34+细胞数的相关性,探索外周血MNC在下肢动脉缺血性疾病治疗中的优势.方法 对59例患者治疗前静息痛、冷感、间歇性跛行及组织损伤分别进行评分,而后动员并采集自体外周血MNC,于缺血下肢多位点等间距注射.评价治疗后第7天及4个月时的治疗效果,分析注入的MNC数及其中所含CD34+细胞数与疗效的相关性.结果 MNC移植后第7天及4个月时患者的下肢缺血均有不同程度的改善,CD34+细胞数量与疗效的相关系数为0.461(P=0.047),尼莫地平值=0.484+1.055×CD34+细胞数,MNC数量与疗效的相关系数为0.473(P=0.000),尼莫地平值=0.288+0.401×MNC数.结果 显示MNC数量比CD34+细胞数量与疗效的相关性更强.结论 注入的自体外周血MNC数较CD34+细胞数更能反映与临床疗效的相关性.

Abstract：

Objective To analyze the efficacy and its correlation with species of transplant cells of autologous mobilized peripheral blood(PB) mononucleated cells (MNCs) transplantation on 59 patients with lower limbs ischemia. Methods Fifty-nine patients were evaluated with symptoms scores and after that their PBMNCs were mobilized and collected and then injected into the ischemic area at equal distance. They effectiveness and scores were evaluated at 7th day and 4th month after therapy. The correlation of CD34 + cells and of MNCs with effectiveness was analysed respectively, and formula for c orrelations between them and effectiveness was calculated. Results After MNCs injection, the effectiveness was observed both at 7th day and 4th month . The correlation of MNCs with effectiveness was stronger than that of CD34 + cells ( the effectiveness was represented by nimodipine value), According to the formula of nimodipine value, the value of the latter =0.484 + 1. 055 × CD34 + cells number and the former = 0.288 + 0. 401 × MNCs number with a correlation coefficient of R =0. 461( P =0. 047 ) and R =0. 473 ( P =0. 000 ) respectively. Conclusion Autologous mobilized PBMNCs number is a better indicator for effectiveness than CD34 + cells number.     

Autologous transplantation of granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells improves critical limb ischemia in diabetes

OBJECTIVE: To assess the application of autologous transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMNCs) in the treatment of critical limb ischemia (CLI) of diabetic patients and to evaluate the safety, efficacy, and feasibility of this novel therapeutic approach. RESEARCH DESIGN AND METHODS: Twenty-eight diabetic patients with CLI were enrolled and randomized to either the transplant group or the control group. In the transplant group, the patients received subcutaneous injections of recombinant human G-CSF (600 microg/day) for 5 days to mobilize stem/progenitor cells, and their PBMNCs were collected and transplanted by multiple intramuscular injections into ischemic limbs. All of the patients were followed up after at least 3 months. RESULTS: At the end of the 3-month follow-up, the main manifestations, including lower limb pain and ulcers, were significantly improved in the patients of the transplant group. Their laser Doppler blood perfusion of lower limbs increased from 0.44 +/- 0.11 to 0.57 +/- 0.14 perfusion units (P < 0.001). Mean ankle-brachial pressure index increased from 0.50 +/- 0.21 to 0.63 +/- 0.25 (P < 0.001). A total of 14 of 18 limb ulcers (77.8%) of transplanted patients were completely healed after cell transplantation, whereas only 38.9% of limb ulcers (7 of 18) were healed in the control patients (P = 0.016 vs. the transplant group). No adverse effects specifically due to cell transplantation were observed, and no lower limb amputation occurred in the transplanted patients. In contrast, five control patients had to receive a lower limb amputation (P = 0.007, transplant vs. control group). Angiographic scores were significantly improved in the transplant group when compared with the control group (P = 0.003). CONCLUSIONS: These results provide pilot evidence indicating that the autologous transplantation of G-CSF-mobilized PBMNCs represents a simple, safe, effective, and novel therapeutic approach for diabetic CLI.     

G-CSF-mobilized peripheral blood mononuclear cells from diabetic patients augment neovascularization in ischemic limbs but with impaired capability

BACKGROUND: Autologous transplantation of mobilized peripheral blood mononuclear cells (M-PBMNCs) is a novel approach to improve critical limb ischemia (CLI) in diabetes. However, endothelial progenitor cells (EPCs) from diabetes are dysfunctional and impaired in ischemia-induced neovascularization. OBJECTIVE: This study aimed to confirm the compromised efficiency of diabetic M-PBMNCs in therapeutic neovascularization, and to determine the underlying mechanisms of this impairment. METHODS: Diabetic M-PBMNCs from 17 diabetic patients or healthy controls, or phosphate-buffered saline (PBS) were injected into the ischemic limbs of streptozotocin-induced diabetic nude mice. The limb blood perfusion, ambulatory score, ischemia damage, capillary/fiber ratio, arteriole density, collateral vessel formation, and pericytes recruitment were evaluated between these three groups. Non-invasive real time image and histopathology were used to detect the in vivo role of transplanted M-PBMNCs. Proliferation and adhesion of EPCs were assayed. In vitro vascular network incorporation and matrigel plug assay were used to test the pro-neovascularization role of M-PBMNCs. RESULTS: Transplantation of diabetic M-PBMNCs also improved neovascularization, but to a lesser extent from that observed with non-diabetic ones. This was associated with the impairment of diabetic M-PBMNCs capacity to differentiate into EPCs, to incorporate into vessel-like tubules in vitro, to participate in vascular-like structure formation in a subcutaneous matrigel plug, and to stimulate the recruitment of pericytes/smooth muscle cells. In addition, there was impairment in vasculogenesis, which was related to the reduced adhesion ability of EPCs from diabetic M-PBMNCs. CONCLUSIONS: Diabetes reduced the capacity of M-PBMNCs to augment neovascularization in ischemia.     

血管内皮生长因子对CD34+干/祖细胞来源的树突细胞分化及功能的影响

目的　探讨血管内皮生长因子 (VEGF)对人脐血CD34 干 /祖细胞来源的树突细胞(dendriticcell,DC)分化和功能的影响。方法　利用免疫磁珠分离法 (MACS)分离纯化脐血CD34 造血干 /祖细胞 ,并在体外将其诱导扩增为DC ,观察VEGF在培养早期和晚期对DC分化和功能的影响。观察培养过程中细胞增殖方式 ,用流式细胞术检测DC表面分化相关抗原CD1α、CD83、CD80、CD5 4、HLA DR等的表达 ,混合淋巴细胞反应法测定DC体外刺激同种异体T细胞增殖的能力 ,ELISA法检测DC培养上清中IL 12的含量。结果　在细胞增殖方面 ,培养第 1天加入VEGF(2 5ng/ml)可显著促进细胞增殖 ,第 14天收获的总细胞数量较对照组增高 (1.5 1± 0 .2 3)倍 (P =0 .0 0 1) ,而培养第 9天加入VEGF则未出现明显的促细胞增殖效应 (P >0 .0 5 ) ;在细胞分化和功能方面 ,培养第 1天加入VEGF明显抑制DC的分化和功能 ,第 1天加VEGF组和对照组DC分化抗原的表达CD1a分别为(33.0 0± 2 .12 ) %和 (81.2 0± 6 .93) % ,CD83分别为 (42 .2 3± 1.15 ) %和 (87.98± 9.79) % ,CD80分别为 (42 .93± 1.32 ) %和 (94 .5 3± 0 .87) % ,HLA DR分别为 (37.93± 5 .30 ) %和 (74 .15± 3.74 ) % (P值均 <0 .0 0 1) ,同时CD14的表达较对照组明显升高 ;刺激同种异体T淋巴     

外周血内皮祖细胞移植对肢体缺血的改善作用

背景:内皮祖细胞移植为肢体缺血的治疗提供了新的选择。目的:研究人外周血来源内皮祖细胞移植对改善肢体缺血的作用。方法:采用Ficoll密度梯度离心法获取人外周血单个核细胞,体外诱导扩增6d后,检测其内皮祖细胞特异性标志的表达,并将荧光染料标记后的贴壁细胞通过缺血局部多点注射移植到后肢缺血的裸鼠动物模型体内,以评价其治疗效果。结果与结论:从人外周血单个核细胞诱导出的贴壁细胞可表达内皮祖细胞特异性标志物CD133、CD34和血管内皮生长因子受体2,说明从人外周血单个核细胞中可诱导出内皮祖细胞。移植内皮祖细胞后裸鼠缺血后肢的坏死情况和毛细血管密度均明显改善(P<0.05);在缺血后肢肌肉石蜡切片中可见分散不均的红色和黄绿色荧光标记的内皮祖细胞的掺入。表明移植的内皮祖细胞可以定向整合到缺血局部,改善裸鼠的后肢缺血。     

自体外周血干细胞移植治疗下肢动脉硬化性闭塞症

目的评价动员后自体外周血干细胞移植治疗下肢动脉硬化性闭塞症(ASO) 的临床疗效及部分影响因素.方法确诊严重ASO患者1例,予rhG-CSF 300μg皮下注射,每日2次,进行外周血干细胞动员,第5天用血细胞分离机单采干细胞,配成干细胞混悬液.将干细胞混悬液肌肉注射进行双下肢移植(3×109细胞/肢).44 d后给病情较重的左下肢第2次移植相同的细胞数.观察3个月,进行各项指标综合评估.结果外周血干细胞移植后,患者疼痛、患肢冷感、间歇性跛行、溃疡明显好转,左右足踝压指数(ABI)分别由0.49,0.69升为0.50,0.85.移植后1个月末梢血流波幅[(11.90±10.95)mm]和激光多谱勒扫描血流灌注量[(0.49±0.16)PU]较移植前[分别为(1.70±1.95)mm,(0.27±0.08)PU]显著改善(P<0.01). 数字减影下肢动脉造影结果显示新侧支血管形成明显增加(评分结果为3).未出现并发症和不良反应.结论采用动员后的外周血干细胞移植治疗ASO患者,方法简单、安全、有效,值得推广应用和深入研究.     

Rapid and effective CD3 T-cell depletion with a magnetic cell sorting program to produce peripheral blood progenitor cell products for haploidentical transplantation in children and adults

BACKGROUND: Effective T-cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting-based direct large-scale T-cell depletion, which is an attractive alternative to standard PBPC enrichment procedures. STUDY DESIGN AND METHODS: PBPCs were harvested from 11 human leukocyte antigen (HLA)-haploidentical donors. T cells labeled with anti-CD3-coated beads were depleted with a commercially available magnetic separation unit (CliniMACS, Miltenyi Biotec) with either the Depletion 2.1 (D2.1, n=11) or the novel Depletion 3.1 (D3.1, n=12) program. If indicated, additional CD34+ selections were performed (n=6). Eleven patients received T-cell-depleted grafts after reduced-intensity conditioning. RESULTS: The median log T-cell depletion was better with the D2.1 compared to the D3.1 (log 3.6 vs. log 2.3, p<0.05) and was further improved by introducing an immunoglobulin G (IgG)-blocking step (log 4.5 and log 3.4, respectively). The D3.1 was superior to the D2.1 (p<0.05) in median recovery of CD34+ cells (90% vs. 78%) and in median recovery of CD3- cells (87% vs. 76%). The median processing times per 10(10) total cells were 0.90 hours (D2.1) and 0.35 hours (D3.1). The transplanted grafts (directly T-cell-depleted products with or without positively selected CD34+ cells) contained a median of 10.5 x 10(6) per kg CD34+, 0.93x10(5) per kg CD3+, and 11.6x10(6) per kg CD56+. Rapid engraftment was achieved in 10 patients. The incidences of acute graft-versus-host disease were less than 10 percent (Grade I/II) and 0 percent (Grade III/IV). CONCLUSION: The novel D3.1 program with IgG blocking enables highly effective, time-saving large-scale T-cell depletion. Combining direct depletion techniques with standard CD34+ selection enables the composition of grafts optimized to the specific requirements of the patients.     

Human umbilical cord blood-derived CD34+ cells may attenuate spinal cord injury by stimulating vascular endothelial and neurotrophic factors

Human umbilical cord blood-derived CD34(+) cells were used to elucidate the mechanisms underlying the beneficial effects exerted by cord blood cells in spinal cord injury (SCI). Rats were divided into four groups: (1) sham operation (laminectomy only); (2) laminectomy + SCI + CD34(-) cells (5 x 10(5) human cord blood lymphocytes and monocytes that contained <0.2% CD34(+) cells); (3) laminectomy + SCI + CD34(+) cells (5 x 10(5) human cord blood lymphocytes and monocytes that contained approximately 95% CD34(+) cells); and (4) laminectomy + SCI + saline (0.3 mL). Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. CD34 cells or saline was administered immediately after SCI via the tail vein. Behavioral tests of motor function measured by maximal angle an animal could hold to the inclined plane were conducted at days 1 to 7 after SCI. The triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis, respectively. To elucidate whether glial cell line-derived neurotrophic factor (GDNF) or vascular endothelial growth factor (VEGF) can be secreted in spinal cord-injured area by the i.v. transplanted CD34(+) cells, analysis of spinal cord homogenate supernatants by specific enzyme-linked immunosorbent assay for GDNF or immunofluorescence for VEGF was conducted. It was found that systemic administration of CD34(+), but not CD34(-), cells significantly attenuated the SCI-induced hind limb dysfunction and spinal cord infarction and apoptosis. Both GDNF and VEGF could be detected in the injured spinal cord after transplantation of CD34(+), but not CD34(-), cells. The results indicate that CD34(+) cell therapy may be beneficial in reversing the SCI-induced spinal cord infarction and apoptosis and hindlimb dysfunction by stimulating the production of both VEGF and GDNF in a spinal cord compression model.