混合脐血血浆对脐血CD34^＋细胞体外扩增的作用

采用两步法分离出脐血CD34~ 细胞,比较研究了混合脐血血浆联合IL-3,IL-6,GM-CSF,Epo 4种中、晚期造血因子和单纯的4种造血因子情况下脐血CD34~ 细胞的体外扩增。结果表明,混合脐血血浆联合造血因子对粒-巨噬细胞集落形成单位(granulocyte-macrophage colony-forming unit,CFU-GM),红系爆式集落形成单位(burst-forming unit of erythriod,BFU-E),混合集落形成单位(minxed colony-forming unit,CFU-mix)3种集落的扩增效果明显优于单纯的4种造血因子联合组,差异具有统计学意义(P<0.01),但单纯混合脐血血浆扩增效果较差。脐血造血细胞扩增对子成人脐血移植有重要意义。上述结果提示,混合脐血血浆的扩增成功,可代替或弥补早期造血生长因子的作用,用于脐血造血细胞的体外扩增。     

IL—6基因转染的骨髓基质细胞系对骨髓移植小鼠造血功能恢复？…

目的：探讨白细胞介素６（ＩＬ－６）基因转染的骨髓基质细胞系ＱＸＭＳＣ１ＩＬ－６对骨髓移植后造血功能的重建作用。方法：将骨髓造血细胞和骨髓基质细胞系一起经尾静脉注射给同系小鼠，建立骨髓移植（ＢＭＴ）模型。小鼠的造血功能用脾结节（ＣＦＵ－Ｓ）、粒－单系祖细胞（ＣＦＵ－ＧＭ）、红系祖细胞（ＣＦＵ－Ｅ、ＢＦＵ－Ｅ）测定及外周血各项血液学指标来确定。结果：ＷＸＭＳＣ１ＩＬ－６转基因骨髓基质细胞可明显增强ＢＭ     

Hematologic aspects of myeloablative therapy and bone marrow transplantation

The transplantation of bone marrow cells or isolated hematopoietic stem cells from the bone marrow or peripheral blood is a widely utilized form of therapy for patients with incurable diseases of the hematopoietic and immune systems. Successful engraftment of the transplanted stem cells in an adequately prepared recipient normally leads to bone marrow reconstitution over a period of several weeks, accompanied by more gradual reconstitution of the immune system. Since the recipient is profoundly ill during the initial treatment period, laboratory data is critical for monitoring engraftment, detecting residual/recurrent disease, and identifying problems that may delay bone marrow reconstitution or lead to other medical complications. Accurate blood cell counts are imperative, and most bone marrow transplantation patients undergo periodic monitoring with bone marrow aspirates and biopsies with cytogenetic, molecular, and multiparametric flow cytometric studies. The potential complications of bone marrow transplantation include engraftment failure and delayed engraftment, infection, residual bone marrow disease, acute and chronic graft versus host disease, myelofibrosis, therapy-related acute leukemia, post-transplant lympho-proliferative disorders, and toxic myelopathy.     

人脐血细胞在SCID小鼠体内生长特性的研究

本实验将人脐血和骨髓单个核细胞,分别经腹腔输入C.B-17SCID小鼠体内,动态观察了受体内人CD3抗原表达及增殖能力。结果输入脐血或骨髓单个核细胞后,受体内均有逐渐增加的人CD3细胞(分别从2.0％、1.5％增至6.8％、5.4％),且脐血组高于骨髓组。接种后60d,二组实验小鼠骨髓CFU-GM产率明显高于对照组(P<0.01),脐血组高于骨髓组(P<0.05)。在60d时受体小鼠外周血、骨髓、肝、脾、肺组织和骨髓CFU-GM集落,用PCR方法检测出人Y染色体特异DNA片段。结果表明,输注人脐血或骨髓细胞到SCID小鼠腹腔后,可在体内长期生长且具有增殖能力。这种模型的建立为今后造血细胞体内研究提供了一种良好途径,也进一步以动物实验证实脐血造血干/祖细胞具有不同于骨髓造血细胞的生物学特性。     

IL-6基因转染的骨髓基质细胞系对骨髓移植小鼠造血功能恢复的促进作用

目的:探讨白细胞介素6(IL-6)基因转染的骨位基质细胞系QXMSC, IL6对骨髓移植后造血功能的重建作用。方法:将骨髓造血细胞和骨髓基质细胞系一起经尾静脉注射给同系小鼠,建立骨髓移植(BMT)模型。小鼠的造血功能用脾结节(CFU-S)、粒-单系祖细胞(CFU-GM)、红系祖细胞(CFU-E、BFU-E)测定及外周血各项血液学指标来确定。结果:QXMSC_1 IL-6转基因骨髓基质细胞可明显增强BMT小鼠形成的CFU-S、CFU-GM、CFU-E和BFU-E数,促进外周血象的恢复。结论:QXMSC_1 IL-6细胞可明显促进小鼠骨髓移植后早期造血功能重建。     

非血缘脐血移植治疗成年人恶性血液病的展望

近年来,非血缘脐血移植(UCBT)治疗成年人恶性血液病取得较多进展.UCBT治疗急性白血病的疗效与单倍体移植、非血缘骨髓移植、外周血干细胞移植的疗效相当,成为治疗恶性血液病的重要选择.单份UCBT的优势在于移植物抗宿主病(GVHD)发生率低且易于控制,保留有较好的移植物抗肿瘤(GVT)效应,并且患者接受治疗后生活质量较好.而UCBT的劣势在于其脐血干细胞数量少(尤其对于成年患者),因此其植入率低、植入较慢、免疫重建慢、移植早期感染发生风险高.改善UCBT的植入率低、免疫重建慢等问题,可以提高UCBT治疗成年人恶性血液病的疗效,丰富造血干细胞移植的供源.本文对今后UCBT治疗成年人恶性血液病,主要在解决UCBT植入问题、免疫重建问题、脐血人类白细胞抗原(HLA)配型等方面进行展望.     

单倍体相合造血干细胞联合脐带血间充质干细胞移植治疗急性重型再生障碍性贫血的疗效观察

本研究探讨单倍体相合造血干细胞移植联合脐带血间充质干细胞治疗重型再生障碍性贫血(SAA)的方法和疗效。对5例SAA的患者进行了单倍体相合造血干细胞移植。移植物选择单倍体相合供者骨髓或外周造血干细胞加脐带血间充质干细胞。观察移植后临床造血重建时间及近期并发症。结果显示,所有SAA患者移植后均获得造血重建,白细胞计数大于2×109/L的平均时间是13.8天,血小板计数大于20×109/L的平均时间是17.8天,第30天行患者外周血STR-PCR检测显示为完全供者的基因型。除1例发生癫痫失去联系外,其余4例均无病存活至今,仍在继续随访中。总之,单倍体相合造血干细胞联合脐带血间充质干细胞移植是治疗急性SAA有效可行的方法,但还须大样本的研究。     

Fanconi anemia--genotoxic stress and senescence of hematopoietic stem cells

Fanconi anemia (FA) is a genetically heterogeneous inherited disorder characterized by progressive bone marrow failure, development of hematopoietic and solid malignancies and genomic instability. 13 FA proteins, identified to date, closely cooperate with familial breast cancer susceptibility proteins such as BRCA2 and PALB2, thereby forming 'the FA/BRCA molecular network'. Here I summarize our recent understanding of the molecular network and its significance in the pathogenesis of FA. I emphasize that FA provides an excellent genetic model for studying senescence and malignant transformation of human hematopoietic stem cells.     

LAK细胞和活化NK细胞对小鼠骨髓造血功能影响的实验研究

目的 探讨淋巴细胞因子激活的杀伤细胞／ 活化自然杀伤细胞（ＬＡＫ 细胞／ 活化ＮＫ 细胞）与造血的关系及其对同基因骨髓移植小鼠造血重建的影响。方法 ①将同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞与骨髓细胞一起置于体外半固体琼脂培养体系中,观察其对ＣＦＵＧＭ 形成的影响。②在小鼠同基因骨髓移植中, 联合注射同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞, 观察其对移植后小鼠造血重建的影响。结果①当骨髓细胞置于有外源性集落刺激因子（ＣＳＦ） 的适宜培养体系中,同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞抑制ＣＦＵＧＭ 形成;而当骨髓细胞置于无ＣＳＦ 不良培养体系中,同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞促进ＣＦＵＧＭ 形成。②在小鼠同基因骨髓移植中,联合注射同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞,能显著促进ＣＦＵＳ 和ＣＦＵＧＭ 增殖和显著升高白细胞和血小板数量。结论 ①同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞对ＣＦＵＧＭ 形成有双向调节作用。②在小鼠同基因骨髓移植中,联合注射同基因ＬＡＫ 细胞／ 活化ＮＫ 细胞,能促进移植后小鼠造血重建。     

Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC).

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.     

Peripheral blood stem cell transplantation for breast cancer patients with bone marrow metastases using GM-CSF priming

Twenty-seven patients with metastastic breast cancer to the bone marrow underwent successful collection of peripheral blood progenitor cells (PBPC) following GM-CSF cytokine priming and were engrafted following courses of high-dose chemotherapy. Myeloid engraftment was observed in a median of 12 days, with a range of 8-29 days. The cell dose infused correlated, although weakly, with days to engraftment, although assays of CFU-GM and CD34+ cells did not, suggesting refinement in such assays is needed. The failure to observe complete remission of the tumor suggests alternative chemotherapy regimens should be investigated.     

Cellular therapies supplement: strategies for improving transplant efficiency in the context of cellular therapeutics

The field of hematopoietic stem cell transplantation (HSCT) has overcome many obstacles that have led to our current clinical ability to utilize cells collected from marrow, mobilized peripheral blood, or umbilical cord blood for the treatment of malignant and nonmalignant hematologic diseases. It is in this context that it becomes evident that future progress will lie in our development of an understanding of the biology by which the process of HSCT is regulated. By understanding the cellular components and the mechanisms by which HSCT is either enhanced or suppressed it will then be possible to design therapeutic strategies to improve rates of engraftment that will have a positive impact on immune reconstitution post-HSCT. In this review we focus primarily on allogeneic hematopoietic stem cell transplantation (allo-HSCT), the current challenges associated with allo-HSCT, and some developing strategies to improve engraftment in this setting.     

HLA不全相合非亲缘供者脐血移植治疗二例儿童急性淋巴细胞白血病

目的　探讨HLA不全相合非亲缘供者脐血移植 (CBT)治疗血液系统恶性肿瘤造血及免疫重建情况及移植相关并发症。方法　对 2例儿童急性淋巴细胞白血病 (ALL)患儿进行HLA 1个位点不合非亲缘供者的CBT。预处理方案 :例 1采用白消安、环磷酰胺 (BU CTX)方案 ,例 2采用BU CTX+卡氮芥方案。移植物抗宿主病 (GVHD)的预防采用环孢菌素A(CsA) +霉酚酸酯 (MMF)方案。移植有核细胞数 (NC)例 1为 14.6× 10 7 kg ,例 2为 16 .2 4× 10 7 kg ,CD3 4+细胞例 1为 7.2 4× 10 5 kg ,例 2为2 1.11× 10 5 kg。结果　例 1和例 2中性粒细胞绝对计数 >0 .5× 10 9 L的时间分别为移植后第 5天和第7天 ;血小板计数 >5 0× 10 9 L的时间分别为移植后第 5 3天和第 46天 ;全血细胞恢复正常的时间分别为移植后第 6 0天和第 5 2天。例 1、例 2分别于移植后第 6 0天和第 30天免疫功能开始恢复 ,移植后第134天和第 12 2天免疫功能恢复正常。移植后第 19天和第 17天DNA指纹图提示供者型。例 1供者为男婴 ,移植后第 49天外周血及骨髓染色体检查均为供者型 :46 ,XY ,10 0 %嵌合。例 1和例 2分别于移植后第 2 6天和第 2 1天出现Ⅱ度急性GVHD。 2例受者已无病生存 2 1个月和 16个月余。结论　CBT造血重建与免疫重建快而稳定 ,移植相关并发症     

人类脐血中多向祖细胞的体外无血清培养及其增殖能力的研究

本文应用造血细胞体外无血清培养技术,对脐血中的CFU-GM,CFU-GEMM,BFU-E进行培养,并与成人骨髓、外周血对照。结果显示:脐血中的CFU-GEMM为成人外周血中的5～8倍,相当于骨髓的70％左右。本文同时用白细胞介素-3(IL-3)将骨髓、外周血和脐血中的单个核细胞孵育3天后,再测定CFU-GEMM,以比较不同来源的祖细胞增殖能力,结果表明,脐血中的多向祖细胞增殖能力最强。     

脐血复温后细胞输入剂量对植入的预测作用分析

本研究探讨非血缘供者脐带血移植(unrelated cord blood transplantation,UCBT)中复温后供者细胞输入剂量对植入和造血重建的预测作用。回顾性分析1999年8月至2010年4月间接受单份UCBT的97例儿童恶性及非恶性疾病患者的临床资料,比较冷冻前和复温后检测的总有核细胞(total nucleated cells,TNC)、CD34+细胞及粒-巨噬细胞集落形成单位(colony-forming unit-granulocyte-macrophage,CFU-GM)的输入剂量对受者的植入及造血重建速度的影响,供者脐带血均来自广州脐血库。结果表明,冷冻前TNC(/kg)(mean±SD:7.65×107±4.26×107;median:6.34×107)、CD34+细胞(/kg)(mean±SD:4.64×105±4.47×105;median:3.03×105)及CFU-GM(/kg)(mean±SD:0.79×105±1.09×105;median:0.57×105)与其对应的复温后TNC(/kg)(mean±SD:6.98×107±4.12×107;median:6.00×...     

小鼠骨髓腔内输注人脐血造血干/祖细胞植活水平的观察

目的 探讨小鼠骨髓腔内输注能否增强人脐血造血干/祖细胞(HS/PC)异种移植的植活能力.方法 将不同数量(1×103、1×104、0.5×105、1×105、5×105)的人脐血CD34+细胞经尾静脉和骨髓腔途径移植入经亚致死剂量照射的NOD/SCID小鼠.于指定时间点处死小鼠,用PCR法检测人17号染色体α-微卫星特异性片段及用流式细胞术检测人源CD45+细胞,观察脐血CD34+细胞在移植小鼠左、右两侧胫骨和股骨及脾脏等部位的归巢及其长期植活能力.结果 异种移植后24h,经骨髓腔移植5×105 CD34+细胞的小鼠肝、脾、肺组织,外周血,左右两侧胫骨和股骨、骨髓细胞均表达人17号染色体α-卫星特异性片段.不同数量的人脐血CD34+细胞经骨髓腔途径移植入NOD/SCID小鼠后,8周时其植活良好(检测部位包括输注部位右侧胫骨,以及非输注部位右侧股骨、左侧胫骨、左侧股骨、脾脏及外周血).分别经尾静脉和骨髓腔两种途径输注同一来源的人脐血CD34+细胞1.0×105,8周时两组间人造血细胞植活水平分别为(44.063±20.095)%和(45.881±22.316)%,差异无统计学意义(P＜0.05);而将移植CD34+细胞数降至1.0×104时,则经骨髓腔途径输注的人脐血CD34+细胞小鼠植活水平(54.019±31.338)%显著优于尾静脉输注途径[(12.197±10.350)%,P＜0.01)];当CD34+细胞输注量降至1.0×103时,仅有骨髓腔内输注组小鼠能见到人脐血CD34+细胞植活,且植活部位通常为非输注部位骨骼.结论 小鼠骨髓腔内移植能够增强人脐血造血干/祖细胞的植活水平.     

A simplified approach to improve the efficiency and safety of ex vivo hematopoietic gene therapy in fanconi anemia patients

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by genetic instability of cells lacking a functional FA/BRCA pathway. Previous studies have shown that in vitro stimulation of bone marrow cells (BMCs) from FA mice promotes apoptosis, reduces the reconstitution ability of the stem cells, and induces myelodysplasia and myeloid leukemia upon reinfusion of the cells. This suggests the convenience of adapting standard protocols of gene therapy to FA. Here we show that the reserve of BM progenitors in FA patients is generally below 20% of normal values. Because this reduced reserve could activate the cycling of BM progenitors, we developed a simplified protocol to transduce BMCs from FA patients with gammaretroviral vectors. We demonstrate that a short in vitro manipulation (12-24 hr) of fresh mononuclear BMCs is sufficient to transduce 42% of hematopoietic progenitors from FA-A patients, in the absence of in vitro prestimulation. When FANCA-expressing vectors were used, this simple procedure reversed the phenotype of the BM progenitors from these patients. We propose that our approach will be more efficient and safer compared with standard gene therapy protocols for FA.     

小鼠脐血联合骨髓c-kit(+)细胞移植加快造血重建的研究

本研究探讨小鼠脐血与骨髓造血祖细胞联合移植以加快移植后早期造血重建作用,并达到脐血来源细胞长期嵌合的程度。采用MACS间接免疫分选法纯化BDF1小鼠骨髓中lin-sca-1-c-kit+(c-kit+)及lin-sca-1+(sca-1+)细胞群,用半固体集落培养结合细胞形态学方法比较二者的体外生物学特性。将60Coγ射线照射的BDF1小鼠(CD45.2)作为受体鼠;接受来自同基因小鼠(CD45.1)骨髓c-kit+细胞群与DBA/2新生小鼠外周血(NPB)的联合移植作为实验组,以NPB干细胞单独移植以及其与sca-1+细胞的联合移植作为对照组,比较移植后22天内的白细胞及血小板变化。用流式细胞术检测移植60周内受鼠外周血粒细胞、T细胞、B细胞中供、受鼠来源细胞比例的动态变化。结果显示:①与sca-1+细胞比较,c-kit+细胞胞体大,胞浆多,核仁明显,其体外集落体积明显小于sca-1+组,未见高增殖潜能集落(HPP)形成;②与NPB干细胞单独移植比较,当NPB干细胞与1×104、2.5×104或5×104细胞量的c-kit+或sca-1+细胞联合移植时,可使白细胞>1×109/L及血小板>100×109/L所需恢复时间从17天提前至12天;③联合c-kit+细胞的移植实验组至移植后60周时脐血来源粒细胞、B细胞和T细胞分别为(96.68±2.68)%、(92.55±3.04)%和(67.96±7.91)%,比第2周时有明显增加(p<0.01),与此相反,联合sca-1+细胞的移植对照组来自同基因供髓和残髓的细胞逐渐增多,而脐血来源细胞逐渐减少,至移植后60周时,脐血来源的粒细胞,B细胞和T细胞仅占(28.46±18.98)%、(17.75±13.17)%、(6.19±7.62)%,比第2周时明显减少(p<0.01)。结论:脐血与受鼠同基因骨髓c-kit+细胞联合移植可加快移植后早期造血恢复,并达到粒细胞、B细胞、T细胞中脐血来源细胞的长期完全或主要嵌合。     

两种第三方细胞联合单倍体相合造血干细胞移植治疗慢性再生障碍性贫血1例

本研究观察脐带间充质千细胞（mesenchymalstemcell,MSC）和单倍体相合脐血细胞作为第三方细胞联合单倍体相合异基因造血干细胞移植治疗慢性再生障碍性贫血的安全性和疗效。患者为女性,12岁,诊断为慢性再生障碍性贫血11年,供者为其母亲,单倍体相合,移植物为经粒细胞集落刺激因子（granulocytecolony-stimulatingfactor,G-CSF）动员的骨髓以及外周血追血干细胞。同时加入分离、扩增的hUC-MSC及患者胞弟HLA单倍体相合脐血细胞作为第三方细胞联合移植。移植物抗宿主病（graft-versus-hostdisease,GVHD）预防采用环孢菌素A＋ATG＋霉酚酸酯＋短程甲氨喋呤＋CD25单克隆抗体。结果表明：输注供者的MNC总数和CD34细胞数分别为7．92×10^8／L和3．78×10^6／L,中性粒细胞大于0．5×10^9／L和血小板大于20×10^9／L的时间分别为12d和14d,35d嵌合体94％,无严重并发症出现。结论：从初步结果看来,联合两种第三方细胞的单倍体相合异基因造血干细胞移植治疗CAA安全、疗效令人满意。     

不同类型异基因造血干细胞移植后的急性白血病患者骨髓形态学比较

目的观察单体型相合造血干细胞移植及全相合异基因造血干细胞移植后骨髓形态学差异及造血重建情况。方法在235名实施异基因造血干细胞移植患者中,单体型相合120名,采用外周血造血干细胞联合骨髓移植;115名全相合的患者,采用单纯的外周血造血干细胞移植。在造血干细胞移植成功出层流室+30 d行骨髓穿刺抽吸涂片观察骨髓形态学差异,并观察造血重建情况。结果 235名患者均成功重建造血。全相合患者白细胞及血小板重建的时间分别14.7 d(12～26 d)及20.6 d(13～32 d);单体型相合造血干细胞移植患者白细胞及血小板重建的时间分别12 d(10～21 d)及18 d(15～30 d);2者之间的差异具有统计学意义(P0.05)。2组患者造血干细胞移植后骨髓全部完全缓解。在骨髓增生程度、粒红比例和细胞形态等方面有差异,但差异无统计学意义(P0.05)。结论单体型相合造血干细胞移植较全相合移植造血重建早,移植后骨髓形态学有差异,但差异无统计学意义。