韩国脐血移植和脐血库的现状（附视频）

自1996年韩国完成首例脐血移植后,各大医院陆续开展了该项技术并建立起脐血库。本讲座介绍了韩国儿童脐血移植的现状、静脉输注冻存的自体脐血单个核细胞改善脑瘫儿童神经功能的试验以及韩国脐血库建设情况和脐血移植的选择策略。     

脐血造血干细胞移植治疗急性淋巴细胞白血病

20 0 0年 3月我们成功地应用人类白细胞抗原 (ＨＬＡ)相合的同胞脐血移植治疗 1例急性淋巴细胞白血病患儿 ,现总结如下。一、患者资料患者 ,男 ,5岁半。骨髓检查为急性淋巴细胞白血病 ,免疫分型为Ｔ淋巴细胞型 ,染色体检查未发现异常 ,术前骨髓涂片检查为ＡＬＬ Ｌ2完全缓解象 ,外周血象无异常。肝脾未触及 ,肝肾功能正常 ,巨细胞病毒 (ＣＭＶ)ＩｇＭ均为阴性。二、移植方法1.脐血的采集及冻存 :2 0 0 0年 2月 3日为孕 37 2ｗ的患者母亲行剖宫产术 ,断脐后采集脐血共 83ｍｌ。采用6 %羟乙基淀粉 (ＨＥＳ) ,两次离心法分离脐血有核细胞 ,去除…     

非血缘脐血造血干细胞移植治疗恶性血液病50例临床观察

目的 回顾性分析非血缘脐血移植(UCBT)治疗恶性血液病患者的植入、移植相关并发症及存活情况.方法 2000年4月至2009年8月,单中心进行UCBT治疗恶性血液病患者50例,其中高危难治性患者39例(78%),中位年龄19岁(3～48岁),中位体重53 kg(15～76 kg).进行单份脐血移植24例,双份脐血移植26例.移植前采用清髓性预处理方案45例,减低强度预处理方案5例.采用环孢素A(CsA)联合吗替麦考酚酯(MMF)预防移植物抗宿主病(GVHD).结果 全部受者按自身的体重计算,输注的脐血总有核细胞数(TNC)中位值为4.0(1.95～16.24)×10~7/kg,其中CD34~+细胞数为2.74(0.67～29.28)×10~5/kg.50例受者中,除1例移植后16 d死亡外,可评估的49例中有42例获得稳定造血功能重建,植入率为86%.中性粒细胞绝对计数(ANC)≥0.5×10~9/L和血小板≥20×10~9/L的中位时间分别为移植后19 d和34 d,移植后42 d时,中性粒细胞恢复率为86.3%(95%可信区间为0.769～0.957).移植后120 d时,血小板恢复率为72.3%(95%可信区间为0.620～0.821).在42例植入的受者中,发生急性GVHD 20例,其中Ⅲ～Ⅳ度3例(发生率为7.1%).移植后2年时,慢性GVHD发生率为17.4%.中位随访时间22个月(4～116个月),移植后180 d时的总存活率为66.2%(95%可信区间为0.590～0.734),1年时的总存活率为57.4%(95%可信区间为0.496～0.652),2年时的总存活率为54.2%(95%可信区间为0.462～0.622).非疾病进展期行移植的受者,180 d时的总存活率为73.2%(95%可信区间为0.659～0.805),1年时的总存活率为66.1%(95%可信区间为0.579～0.743),2年时的总存活率为62.2%(95%可信区间为0.542～0.682).移植后原发病复发5例,2年累计复发率为16.2%(95%可信区间为0.099～0.225).移植后死亡21例,感染为主要死亡原因.结论 UCBT治疗恶性血液病是安全和有效的.     

非清髓性脐血移植治疗成人重型再生障碍性贫血

目的　研究与追踪观察无关供者脐血造血干细胞移植 (Allo CBSCT)治疗成人重型再生障碍性贫血 (SAA)后 ,脐血造血细胞的植入情况及疗效。方法　单份脐血 (3例 )和双份脐血 (3例 )移植治疗成人重型再生障碍性贫血患者共 6例。输入的脐血单个核细胞 (MNC)数为 (1.6～ 10 .7)×10 7/kg(按患者体重计 )。预处理方案由低剂量的环磷酰胺 (CTX)和抗淋巴细胞球蛋白 (ALG)组成。CTX总用量为 6 0mg/kg ;ALG为 12 0mg/kg。移植后供者细胞植入状态的检测方法采用微卫星DNA指纹法或萤光定量PCR分析。结果　 6例中有 5例移植后有供者细胞植入证据 ,均为供、受者细胞混合嵌合状态。 2例接受双份脐血移植者均仅显示单份脐血植入。迄今追踪时间平均 2 1个月 (7～ 5 0个月 ) ,4例造血完全恢复。 2例移植后早期死于重症感染 ,其中 1例曾有早期植入证据。结论　Allo CBSCT治疗成人重型再生障碍性贫血 ,可形成较长期的稳定供、受者造血细胞混合嵌合体。脐血可作为造血干细胞来源的一种选择。     

脐血单个核细胞体外扩增的实验研究

目的 探讨含有细胞因子的无血清培养基对脐血单个核细胞(MNC)体外培养后的扩增情况和用于移植的安全性.方法 从新鲜脐血中分离出的MNC,在含细胞因子的无血清培养体系中培养.分别将培养前和培养第10天时的造血细胞进行细胞计数、细胞活力分析、集落分析、流式细胞仪检测表面标记、彗星试验分析DNA的损伤情况、无菌性分析及移植至NOD/SCID小鼠体内等项研究.结果 经过体外短期培养,脐血中MNC、CD34+、CD133+、CD34+CXCR4+及CD34+ VLA-4+细胞扩增倍数均比培养前增高(P<0.05);半固体培养基可支持多系集落的生长;培养前和培养第10天时脐血细胞DNA损伤率均低于5%;无菌性分析提示细胞未受污染.将体外扩增后的脐血造血细胞移植入NOD/SCID小鼠体内,与新鲜脐血移植相比,小鼠的存活时间及植入能力的差异均无统计学意义(P>0.05).结论 脐血造血细胞体外扩增是解决脐血造血细胞数量不足的有效方法.脐血造血细胞经短期培养能为造血干细胞移植提供安全而具植入能力的造血细胞.     

脐血干/祖细胞的造血潜能和淋巴细胞免疫特性的研究

本文对 3 4份脐血的ＣＤ3 4 细胞数、体外集落形成能力及淋巴细胞表型进行了研究 ,报告如下。一、材料和方法1.脐血的采集 :筛选我院产科无急慢性疾病足月产妇 3 4例。采用密闭式采血法。使用装有 2 8ｍｌ枸橼酸钠葡萄糖保养液 (ＡＣＤ)的 2 0 0ｍｌ无菌采血袋 ,行脐静脉穿刺 ,使脐血由重力作用流入采血袋 ,经常摇动采血袋 ,使脐血与保存液充分混匀。2 .脐血有核细胞 (ＮＣ)的分离 :将采血袋中加入 1/ 4量 60ｇ/Ｌ的羟乙基淀粉氯化钠注射液 ,充分混匀 ,静置 3 0～ 4 5ｍｉｎ ,待细胞分层后弃去红细胞。取少许上层富含ＮＣ悬液 ,供实验使用 …     

双份脐血移植后受者骨髓间充质干细胞嵌合情况

目的 研究双份脐血移植(DCBT)受者骨髓间充质干细胞(MSC)的嵌合状态.方法 急性粒细胞白血病M2a型男性患者1例,接受改良白消安环磷酰胺方案+抗胸腺细胞球蛋白(ATG)预处理,输注5个抗原(5/6)相合和4个抗原(4/6)相合的非血源脐血各1份,移植后19 d粒系造血重建.移植后87 d,采用密度梯度离心法分离DCBT后受者及正常供者的骨髓单个核细胞,分别培养MSC,用流式细胞术检测细胞表面标志,诱导其向成脂肪细胞和成骨细胞分化,应用逆转录聚合酶链反应法检测MSC表面造血及免疫相关分子的表达,短串联重复序列聚合酶链反应检测受者MSC、外周血、骨髓中供者细胞嵌合率.结果 移植后受者MSC与正常供者MSC具有相似的细胞形态、免疫表型以及分化潜能,均能表达白细胞介素6、干细胞因子、白血病抑制因子和粒-巨噬细胞集落刺激因子等造血及免疫相关分子的mRNA.DCBT后受者骨髓优势脐血嵌合度达96.4％,外周血嵌合度达95.7％,MSC的优势脐血嵌合度为5.4％,MSC中受者本身部分占94.6％.结论 DCBT后,受者造血重建仅来自于其中1份脐血.移植后骨髓MSC大部分来源受者本身,部分嵌合的供者MSC来源于植入的单份脐血.     

人脐血内皮祖细胞治疗裸鼠心肌梗死

目的 探讨人脐血内皮祖细胞(EPCs)移植治疗裸鼠心肌梗死的可行性.方法 采用淋巴细胞分离液提取人脐血单个核细胞(MNCs),应用添加诱导因子的培养基于体外诱导分化并于培养7 d后进行鉴定.采用20只裸鼠建立心肌梗死模型后,将体外诱导分化7 d并摄取CM-Dil的内皮祖细胞通过尾静脉注射进行细胞移植到实验组,对照组注射培养基.2周后计数心梗区域新生毛细m管密度及心梗面积并于荧光显微镜下观察新生血管的荧光.结果 体外诱导7 d后贴壁细胞CD34阳性率达(50.48±5.17)%,CDl33阳性率达(19.12±4.37)%.实验组平均梗死面积为(8.27±1.64)%,对照组为(14.30±2.84)%(t=-4.78,P<0.05);实验组每高倍视野平均新生血管密度为14.29±1.38,对照组为10.17±1.72(t=4.71,P<0.01);行荧光显微镜下观察实验组新生血管有红色荧光.讨论人脐血单个核细胞在体外诱导分化为内皮祖细胞,进行细胞移植到建立心梗模型的裸鼠后可在心梗区域形成新生血管,从而并改善梗死部位心脏功能.     

小鼠脐血移植模型研究

一、材料与方法1.材料:选择足月健康产妇,排除ＨＩＶ、ＨＢＶ、ＨＣＶ、梅毒、ＣＭＶ、ＥＢＶ感染,胎儿娩出后断脐,采集脐血备用。2.方法:(1)脐血单个核细胞以羟乙基淀粉(ＨＥＳ)法分离。(2)脐血冻存、复苏:以ＤＭＳＯ20%,人血白蛋白10%,ＲＰＭＩ164070%为保护剂,与细胞悬液以1∶1混合,置4℃30ｍｉｎ,-20℃2ｈ,-40℃4ｈ,液氮气相过夜后,-196℃液氮中保存。使用前置43℃水浴中1ｍｉｎ全融化。以台盼兰拒染检测细胞活力。(3)ＣＦＵＧＭ培养:马血清0.5ｍｌ,ＧＭＣＳＦ10μｌ,ＲＰＭＩ16401.75ｍｌ,30ｇ/Ｌ琼脂0.25ｍｌ,接种细胞数1×106个,37℃…     

人脐血造血干/祖细胞的生物反应器大规模扩增及移植实验

目的 利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法 采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 生物反应器扩增前MNC为(1.2～2.8)×108个,扩增后为(3.7～12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论 利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.     

Optimizing umbilical cord blood collection: impact of obstetric factors versus quality of cord blood units

INTRODUCTION: The main limitation factor for wide use of umbilical cord blood units (UCBs) as a source of hematopoietic progenitors for transplantation is cell dose. International standard guidelines recommend 2 x 10(7)/kg as the minimal nucleated cell dose for UCB transplantation for adults and 3.7 x 10(7)/kg for children. Therefore it is important to the optimize donor selection and the collection method so as to achieve high cell doses. In this study our main purpose was to determine whether obstetric factors influence UCBs collected. STUDY DESIGN: The study involved 304 UCBs collected from January to December 2004. The UCBs were collected after donor selection based on international criteria for cord blood banking. We analyzed UCB biological features such as collected volume, total nucleated cells (TNC), and CD34-positive cells, and obstetric factors. RESULTS: First, our study showed by multivariate analysis that infant weight was the main factor that influenced biologic features of UCB collected such as total volume (P = .000), TNC (P = .000), CD34 total count (P = .003), and CFU-GM (P = .004). Placental weight > 600 g produced a better volume (P = .007) and increased TNC (P = .056). Gestational age > 39 weeks enhanced CD34% (P = .016). Regarding route of delivery, we found that cesarean section produced higher volume and reduced WBC count compared to vaginal delivery, regarding cord length, it increased TNC (P = .037). And last, we noticed that female infants increased WBC (P = .013) and CD34(+) total count (P = .019) more than male ones. CONCLUSIONS: Our results confirm that volume and TNC are influenced by several obstetric factors, such as greater infant and placental weight, predicting a better collection.     

Axonal remyelination by cord blood stem cells after spinal cord injury

Human umbilical cord blood stem cells (hUCB) hold great promise for therapeutic repair after spinal cord injury (SCI). Here, we present our preliminary investigations on axonal remyelination of injured spinal cord by transplanted hUCB. Adult male rats were subjected to moderate SCI using NYU Impactor, and hUCB were grafted into the site of injury one week after SCI. Immunohistochemical data provides evidence of differentiation of hUCB into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Ultrastructural analysis of axons reveals that hUCB form morphologically normal appearing myelin sheaths around axons in the injured areas of spinal cord. Colocalization studies prove that oligodendrocytes derived from hUCB secrete neurotrophic hormones neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF). Cord blood stem cells aid in the synthesis of myelin basic protein (MBP) and proteolipid protein (PLP) of myelin in the injured areas, thereby facilitating the process of remyelination. Elevated levels of mRNA expression were observed for NT3, BDNF, MBP and PLP in hUCB-treated rats as revealed by fluorescent in situ hybridization (FISH) analysis. Recovery of hind limb locomotor function was also significantly enhanced in the hUCB-treated rats based on Basso-Beattie-Bresnahan (BBB) scores assessed 14 days after transplantation. These findings demonstrate that hUCB, when transplanted into the spinal cord 7 days after weight-drop injury, survive for at least 2 weeks, differentiate into oligodendrocytes and neurons, and enable improved locomotor function. Therefore, hUCB facilitate functional recovery after moderate SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.     

Spinal cord blood flow following sub-arachnoid lidocaine

Twelve mongrel dogs were randomized into two equal groups. Cervical, thoracic and lumbosacral spinal cord and spinal dural blood flows were measured using the radioactive microsphere technique. Blood flow determinations were made prior to and 20 and 40 minutes following lumbar subarachnoid injection of: two per cent lidocaine (100 mg) or two per cent lidocaine (100 mg) with 1/25,000 epinephrine (200 micrograms). Dogs receiving subarachnoid lidocaine demonstrated a decrease in mean arterial blood pressure of 23 per cent and 14 per cent (p less than 0.05), while dogs receiving lidocaine with epinephrine had a decrease of 38 and 34 per cent (p less than 0.05) at 20 and 40 minutes respectively. Cardiac index was not significantly changed in either group. Lumbar subarachnoid lidocaine (100 mg) produced a rapid regional dural hyperemia (observed at 20 minutes postinjection) and a delayed regional spinal cord hyperemia (observed at 40 minutes postinjection) which were not observed following the addition of epinephrine (200 micrograms).     

Spinal cord blood flow following subarachnoid tetracaine

Spinal cord and spinal dural blood flow in the cervical, thoracic and lumbosacral regions were measured in dogs using the radioactive microsphere technique. Measurements were taken before and 20 and 40 minutes after lumbar subarachnoid injection of one of the following: physiologic saline; tetracaine 20 mg or tetracaine 20 mg with epinephrine 200 micrograms. No significant change in spinal cord or spinal dural blood flow occurred following subarachnoid physiologic saline or tetracaine with epinephrine. Dogs receiving subarachnoid tetracaine demonstrated a significant increase in lumbosacral spinal cord, and thoracic and lumbosacral dural blood flow at 20 and 40 minutes following injection. Cervical dural blood flow was significantly increased at 40 minutes after subarachnoid tetracaine. Lumbar subarachnoid tetracaine (20 mg) produces a regional spinal cord and generalized dural hyperemia which is prevented by the addition of epinephrine (200 micrograms).     

Glomerular vasculopathy after unrelated cord blood transplantation

A 1-year-old boy with hemophagocytic lymphohistiocytosis exhibited proteinuria 1 month after unrelated cord blood cell transplantation, which persisted without hematuria. Laboratory study showed an increase of factor VIII-related antigen and total plasminogen activator inhibitor, suggesting endothelial injury. Histological examination of autopsy materials showed increased mesangial matrices and double-contoured basement membranes, and ultrastructurally, swelling of the endothelial cells and widening of the subendothelial space with mesangial interposition. Thrombosis was not observed at any of the sites. This case may be vasculopathy distinct from thrombotic microangiopathy (TMA) or a variant form of TMA following blood stem cell transplantation (BSCT). This vasculopathy should be considered in the differential diagnosis of proteinuria in the early stages after BSCT.