IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O^6-METHYLGUANINE-DNA-METHYLTRANS cDNA INTO HUMAN UMBILICAL CORD BLOOD CD34^+ CELLS

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O^6-methylguanine-DNA-methyltrans (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT- PCR method from total RNA of fresh human liven the fragment was cloned into PGEM-T vector and further subcloned into GINa retrovirus vector. Then the GINaMGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8x105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT- cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years-implications for clinical transplantation and stability programs

BACKGROUND Peripheral blood stem cells(PBSC)are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant.Long term cryopreservation is commonly defined as five years or longer,and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft.Clinical programs,stem cell banks,and regulatory and accrediting agencies interested in product stability would benefit from such data.Thus,we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2 Rγnull(NSG)mice.AIM To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units.METHODS PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health.These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice,and the pre-freeze and post-thaw characteristics of the units were compared.Progenitor function was assessed using standard colony-forming assays.CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function.RESULTS Ten PBSC units with mean of 17 years in cryopreservation(range 13.6-18.3 years)demonstrated a mean total cell recovery of 88%±12%(range 68%-110%)and post-thaw viability of 69%±17%(range 34%-86%).BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw.Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units.All mice demonstrated long-term engraftment at 12 wk with mean34%±24%human CD45+cells,and differentiation with presence of human CD19+,CD3+and CD33+cells.Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies.CONCLUSION We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice,signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Effect of Angiotensin Ⅱ on Cord Blood CD^(34+) Cells Expansion in vitro

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34 + cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1±1.8 times to 9 2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

WJSC 6^(th) Anniversary Special Issues(1):Hematopoietic stem cell transplantation Allogeneic hematopoietic cell transplant for acute myeloid leukemia:Current state in 2013 and future directions

Acute myeloid leukemia(AML)represents a heterogeneous group of high-grade myeloid neoplasms of the elderly with variable outcomes.Though remissioninduction is an important first step in the management of AML,additional treatment strategies are essential to ensure long-term disease-free survival.Recent pivotal advances in understanding the genetics and molecular biology of AML have allowed for a risk-adapted approach in its management based on relapse-risk.Allogeneic hematopoietic cell transplantation(allo-HCT)represents an effective therapeutic strategy in AML providing the possibility of cure with potent graft-versus-leukemia reactions,with a demonstrable survival advantage in younger patients with intermediate-or poor-risk cytogenetics.Herein we review the published data regarding the role of allo-HCT in adults with AML.We searched MEDLINE/PubMed and EMBASE/Ovid.In addition,we searched reference lists of relevant articles,conference proceedings and ongoing trial databases.We discuss the role of allo-HCT in AML patients stratified by cytogenetic-and molecular-risk in first complete remission,as well as allo-HCT as an option in relapsed/refractory AML.Besides the conventional sibling and unrelated donor allografts,we review the available data and recent advances for alternative donor sources such as haploidentical grafts and umbilical cord blood.We also discuss conditioning regimens,including reduced intensity conditioning which has broadened the applicability of allo-HCT.Finally we explore recent advances and future possibilities and directions of allo-HCT in AML.Practical therapeutic recommendations have been made where possible based on available data and expert opinion.

Haplo-cord后置模式造血干细胞移植治疗儿童重型再生障碍性贫血的临床分析

目的比较是否辅助第三方脐带血在单倍体造血干细胞移植治疗儿童重型再生障碍性贫血的疗效差异。方法回顾性分析2017年5月至2023年2月上海市儿童医院明确诊断为重型或极重型再生障碍性贫血并接受单倍体造血干细胞移植的38例患儿的临床资料。其中21例患儿接受单倍体造血干细胞联合第三方脐血移植(Haplo-cord HSCT),17例接受单倍体造血干细胞移植(Haplo-HSCT)。均采用清髓性预处理方案:氟达拉滨(Flu)/白消安(Bu)/环磷酰胺(Cy)+兔抗人胸腺细胞球蛋白(rabbit anti-human hymocyte globulin,r-ATG)。结果Haplo-cord组21例患儿均获得造血重建,其中2例发生继发性植入失败。Haplo组中1例患儿原发性植入失败,1例继发性植入失败。Haplo-cord组和Haplo组中性粒细胞植入中位时间分别为12(10,19)和11(10,16)d(P=0.630),血小板植入中位时间为16(12,32)和16(13,24)d(P=0.461)。Haplo-cord组和Haplo组Ⅱ-Ⅳ度急性移植物抗宿主病(acute graft-versus-host disease,aGVHD)、Ⅲ-Ⅳ度aGVHD、慢性移植物抗宿主病(chronic graft-versus-host disease,cGVHD)的累积发生率分别为[(23.8±9.3)%比(25±10.8)%,P=0.770]、[(9.5±6.4)%比(18.7±9.8)%,P=0.374]、[(14.3±7.6)%比(33±12.2)%,P=0.226]。两组患儿3年总体生存率(overall survival,OS)、无失败生存率(failure free survival,FFS)、无GVHD/无失败生存率(GVHD-free and failure-free survival,GFFS)分别为[(95.2±4.6)%比(85.6±9.7)%,P=0.405]、[(83.5±8.9)%比(82.4±9.2)%,P=0.642]、[(81±8.6)%比(52.9±12.1)%,P=0.046]。基于短串联重复序列多态性的STR-PCR嵌合检测提示移植后获得造血重建的患儿均为完全单倍体供者嵌合。我们发现Haplo-cord后置模式治疗儿童重型再生障碍性贫血可获得更高的GFFS。结论单倍体造血干细胞移植在治疗儿童重型再生障碍性贫血方面植入率高,疗效确切,辅助输注脐血可能提高移植的总体疗效。

人类白细胞抗原相合同胞新鲜脐血移植治疗儿童β重型地中海贫血

背景:异基因造血干细胞移植是目前根治重型地中海贫血最有效的方法,然而仅有不到一半的患者能找到相合的骨髓或外周血干细胞。同胞来源脐血干细胞与骨髓和外周血干细胞具有不同的特征,是重型地中海贫血患者移植治疗的一种潜在造血干细胞替代来源。目的:探讨应用人类白细胞抗原相合同胞新鲜脐血移植治疗儿童β重型地中海贫血的疗效。方法:选择2010年6月至2020年6月南方医科大学南方医院收治的β重型地中海贫血患儿48例,男28例,女20例,中位年龄4岁,均接受人类白细胞抗原相合同胞新鲜脐血移植治疗,脐血移植前均采用清髓性不含抗胸腺细胞球蛋白的预处理方案,脐血移植后均采用环孢素A+吗替麦考酚酯+/-短程甲氨蝶呤预防移植物抗宿主病。结果与结论:(1)48例患儿输注的总有核细胞中位数为8.17×10^(7)/kg,输注的CD34^(+)细胞中位数为2.40×10^(5)/kg,脐血移植后中位随访时间98个月,44例植入成功,粒细胞和血小板植入中位时间分别为28 d和31 d,其中37例移植后植入证据检测为供者型完全嵌合,7例移植后早期检测为供受者稳定混合嵌合;(2)44例植入成功患儿中,4例发生急性移植物抗宿主病,分别为Ⅰ级(n=2)和Ⅱ级(n=2),受累器官均为皮肤,均未发生慢性移植物抗宿主病;(3)脐血移植后,48例患儿中5例发生巨细胞病毒感染及激活,12例发生败血症,3例发生侵袭性真菌病,21例发生口腔炎,8例发生出血性膀胱炎,1例发生肝静脉闭塞;(4)48例患儿中,47例存活,1例在移植后28 d因重症肺炎合并急性心力衰竭死亡,43例无病存活,3例发生原发性植入失败,1例发生移植后全血细胞减少,5年总生存率和无病生存率分别为98%和89%,1年时移植相关死亡的累计发病率为2.1%;(5)结果显示,人类白细胞抗原相合同胞新鲜脐血移植治疗儿童β重型地中海贫血的效果良好,移植物抗宿主病的发生风险低。

黑枸杞花青素联合人脂肪源性血管外膜细胞支持脐血造血干/祖细胞的增殖

背景:黑枸杞花青素(Anthocyanins in Lycium ruthenicum Murr,ALRM)是黑枸杞中重要的活性成分之一,具有抗氧化、免疫调节等功效。人脂肪源性血管外膜细胞(CD146^(+)hAD-PCs)是骨髓间充质干细胞的前体细胞,在体外具有促进造血干/祖细胞增殖与分化的功能。ALRM联合CD146^(+)hAD-PCs对脐血造血干/组细胞的体外支持作用有待于研究。目的:探讨ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞体外扩增的支持作用。方法:CCK-8法检测不同质量浓度ALRM(0,200,400,600,800,1000 mg/L)对CD146^(+)hAD-PCs增殖的影响;流式细胞术检测ALRM对CD146^(+)hAD-PCs细胞周期的影响。共培养实验分为空白组、ALRM组、CD146^(+)hAD-PCs组、ALRM+CD146^(+)hAD-PCs组,分析ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞的体外支持作用。共培养1,2,4周,比较扩增后细胞数量、集落形成单位数量,流式细胞仪检测细胞免疫表型,ELISA检测细胞因子水平。结果与结论:(1)ALRM质量浓度为200 mg/L时,CD146^(+)hAD-PCs活力最高,CD146^(+)hAD-PCs的G_(0)/G_(1)期细胞比例下降,S期、G_(2)/M期细胞比例上升(P<0.01)。(2)脐血CD34^(+)造血干/祖细胞数量变化:在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组高于ALRM组(P均<0.05),在共培养2,4周时ALRM+CD146^(+)hAD-PCs组高于CD146^(+)hAD-PCs组(P均<0.05),ALRM组与空白组随着共培养时间延长细胞数量逐渐减少。(3)集落形成能力及免疫表型分析:在共培养1,2周时ALRM+CD146^(+)hAD-PCs组的集落形成单位数量高于CD146^(+)hAD-PCs组和ALRM组(P均<0.05);在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组CD45^(+)、CD34^(+)CD33^(-)细胞比例高于CD146^(+)hAD-PCs组(P均<0.01)。(4)细胞因子变化:在共培养4周时ALRM+CD146^(+)hAD-PCs组的白细胞介素2水平高于ALRM组、CD146^(+)hAD-PCs组(P<0.05);在共培养2,4周时ALRM+CD146^(+)hAD-PCs组白细胞介素3水平高于CD146^(+)hAD-PCs组(P<0.05);在共培养1周时ALRM+CD146^(+)h AD-PCs组的粒细胞集落刺激因子水平高于CD146^(+)hAD-PCs组,在共培养2周时高于ALRM组、CD146^(+)hAD-PCs组(P<0.01);在共培养1,2,4周时ALRM组、ALRM+CD146^(+)hAD-PCs组的干扰素γ水平低于CD146^(+)hAD-PCs组(P<0.05)。(5)由于空白组无基质细胞,脐血CD34^(+)造血干/祖细胞在共培养1周之后就无法计数,未进行免疫表型、集落分析和细胞因子检测。(6)结果表明:ALRM可以通过促进CD146^(+)hAD-PCs增殖和细胞周期转化进而促进脐血CD34^(+)造血干/祖细胞的体外扩增,在造血干细胞移植研究方面具有重要价值。

The impact of HLA haplotype and alleles mismatches of donor-recipient pairs on outcome of haplo-identical hematopoietic stem cell transplantation with a third part cord blood unit

Objective To analyze allele mismatches of HLA-A,-B,-C,-DRB1,-DQB1 and haplotype mismatch of donor-recipient pairs on the outcome of haploidentical transplantation combined with a third part cord blood unit.Methods 230 pairs of donor-recipient were performed HLA-A,B,C,DRB1,DQB1 typing using

Ex vivo expansion of hematopoietic stem cells

Ex vivo expansion of hematopoietic stem cells(HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.

Optimal stem cell source for allogeneic stem cell transplantation for hematological malignancies

Hematopoietic stem cell transplant(HSCT) is a standard treatment for many hematological malignancies.Three different sources of stem cells, namely bone marrow(BM), peripheral blood stem cells(PBSC) and cord blood(CB) can be used for HSCT, and each has its own advantages and disadvantages. Randomized controlled trials(RCTs) suggest that there is no significant survival advantage of PBSC over BM in Human Leukocyte Antigen-matched sibling transplant for adult patients with hematological malignancies. PBSC transplant probably results in lower risk of relapse and hence better disease-free survival, especially in patients with high risk disease at the expense of higher risks of both severe acute and chronic graft-versus-host disease(GVHD).In the unrelated donor setting, the only RCT available suggests that PBSC and BM result in comparable overall and disease-free survivals in patients with hematological malignancies; and PBSC transplant results in lower risk of graft failure and higher risk of chronic GVHD.High level evidence is not available for CB in comparison to BM or PBSC. The risks and benefits of different sources of stem cells likely change with different conditioning regimen, strategies for prophylaxis and treatment of GVHD and manipulation of grafts. The recent success and rapid advance of double CB transplant and haploidentical BM and PBSC transplants further complicate the selection of stem cell source. Optimal selection requires careful weighing of the risks and benefits of different stem cell source for each individual recipient and donor. Detailed counseling of patient and donor regarding risks and benefits in the specific context of the patient and transplant method is essential for informed decision making.

Are globoseries glycosphingolipids SSEA-3 and-4 markers for stem cells derived from human umbilical cord blood?

Umbilical cord blood(UCB)is an efficient and valuable source of hematopoietic stem cells(HSCs)for transplantation.In addition to HSCs it harbours low amounts of mesenchymal stem cells(MSCs).No single marker to identify cord blood-derived stem cells,or to indicate their multipotent phenotype,has been characterized so far.SSEA-3 and-4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells,where SSEA-3 rapidly disappears when the cells start to differentiate.Lately SSEA-3 and-4 have also been observed in MSCs.As there is an ongoing discussion and variation of stem-cell markers between laboratories,we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB.We have performed complementary analysis using gene expression analysis,mass spectrometry and immunochemical methods,including both flow cytometry and immunofluoresence microscopy.SSEA-4,but not SSEA-3,was expressed on MSCs but absent from HSCs.Our findings indicate that SSEA-3 and/or-4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood,as their expression may be altered by cell-culture conditions.

Experimental study on ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG−CSF+rhMDGF combination and the coculture scheme of rhSCF+rhG−CSF+rhMDGF+hMSCs.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5×10^(6) cells per mouse.After transplantation,the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCs,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45~55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.

儿童脐血移植后巨细胞病毒活化的危险因素及其与T细胞重建的相关性

目的 探讨儿童白血病接受脐带血移植后巨细胞病毒(CMV)活化的危险因素及其与T淋巴细胞重建的相关性。方法 回顾性分析2016年1月—2021年12月在苏州大学附属儿童医院血液科行脐带血造血干细胞移植的白血病患儿的临床资料及CMV活化的发生率,并分析单次及多次CMV活化对T淋巴细胞重建的影响,最后随访时间是2022年12月。结果 共纳入75例患儿,移植100d内53例发生CMV活化,18例表现为难治性。单因素分析示:预处理使用ATG是CMV活化的危险因素(P=0.043);移植时HLA配型不合、发生Ⅱ-Ⅳ度aGVHD是难治性CMV活化的危险因素(P<0.01);多因素分析示:发生Ⅱ-Ⅳ度aGVHD是难治性CMV活化的独立危险因素(P=0.021);发生多次CMV活化者移植后1年内CD4^(+)细胞重建延迟。移植后1个月内CMV活化对总体生存率无明显影响,CMV活化的AML患儿累积复发率下降。结论 预处理使用ATG是白血病儿童脐血移植后CMV活化的危险因素,供-受体HLA配型不合、发生Ⅱ-Ⅳ度aGVHD是发生难治性CMV感染的危险因素;多次CMV活化影响CD4^(+)细胞重建。

免疫监控指导治疗脐血移植后急性移植物抗宿主病一例并文献复习

目的探讨以皮疹为主要表现的急性移植物抗宿主病(aGVHD)在免疫监控指导下的精准诊疗效果。方法回顾1例因高危急性髓系白血病接受非血缘脐血移植、其后出现顽固性皮疹的患儿的临床资料及诊治过程,以“造血干细胞移植”“免疫重建”和“急性移植物抗宿主病”的中英文为检索词,对以下数据库的相关论文进行检索:PubMed、Web of Science、CNKI、万方数据知识服务平台,收集检索到的病例资料并进行分析。结果该例1岁9月龄女性患儿成功获得造血重建,移植后22 d患儿头部、后颈部出现密集红色丘疹(面积约19%)、瘙痒明显,口服他克莫司、外用激素类药物后皮疹无好转(面积>90%),此时监测CD3^(+)细胞、CD8^(+)细胞、CD3^(+)CD69^(+)细胞、CD3^(+)HLADR^(+)细胞比例明显升高,调节性T细胞(Treg)比例下降。免疫指标支持T淋巴细胞活化,考虑Ⅱ度aGVHD(皮肤3级),遂予加强免疫抑制治疗方案。期间患儿皮疹有消退,但仍反复,并伴皮肤明显脱屑,复查CD3^(+)细胞、CD8^(+)细胞、CD3^(+)HLA-DR^(+)细胞比例仍偏高,遂再次调整治疗方案、加强抗排斥,患儿皮肤aGVHD好转。定期监测原发病完全缓解、植入比例100%、免疫重建稳定,随访至移植后32个月,患儿无病存活。检索到相关文献9篇,均论证了早期CD3^(+)细胞、CD8^(+)细胞、活化T淋巴细胞比例升高以及Treg下调与aGVHD发生相关。结论对异基因造血干细胞移植术后患者的免疫状态进行动态监测,有利于评估其免疫重建情况及疾病状态(如aGVHD),有助于制定合理的免疫抑制治疗方案,使患者获得良好预后。

脐带血造血干细胞体外生成红细胞过程中高效脱核体系的研究

目的 研究磷脂酰肌醇三羟激酶(PI3K)、组蛋白去乙酰化酶2(HDAC2)和细胞松弛素B对脐带血造血干细胞体外培养促红系脱核的作用,以期建立高效脱核体系。方法 采用磁分选富集脐带血CD34+细胞。在培养第0、4天添加干细胞因子(SCF)、IL-3、促红细胞生成素(EPO),培养第7天添加SCF、EPO,培养第11、15、18天仅添加EPO。分别于第7、11、15和18天添加PI3K(0、25、50、100 ng/mL)、HDAC2(0、60、150、300 ng/mL)、细胞松弛素B(0、25、50、75 ng/μL)3种脱核剂。采用正交设计实验分析3种脱核剂的浓度和添加时间4因素4水平对促红系脱核效果的影响,获得最佳脱核条件并作为优选组,以不加脱核剂培养作为对照组进行验证。细胞培养至第21天时,用流式细胞仪分别检测CD235+、SYTO-64-细胞,计算脱核率。使用血细胞计数仪检测RBC,ELISA法检测2,3-二磷酸甘油(2,3-DPG)、化学发光法检测ATP,观察细胞形态。结果 最佳脱核条件为培养第15天添加PI3K浓度为100 ng/mL、HDAC2浓度为150 ng/mL和松弛素B浓度为75 ng/μL。培养至第21天,优选组红细胞脱核率为(74.30±5.59)%,对照组为(28.30±14.10)%,优选组高于对照组(t=9.099,P=0.012)。培养体系获得RBC平均可达2×1010/L,红细胞ATP、2,3-DPG与正常红细胞无差异,红细胞形态与正常红细胞一致。结论 以上最佳脱核条件建立的培养体系可促进造血干细胞体外生成红细胞高效脱核。

Hematopoietic stem cell transplantation for children with β-thalassemia major: multicenter experience in China

Backgroundβ-Thalassemia major (β-TM) has become a public health problem in China's Mainland. Hematopoietic stem cell transplantation (HSCT) has remained the only cure forβ-TM in China's Mainland since 1998. Methods This multicenter retrospective study provides a comprehensive review of the outcomes of 50 pediatric patients withβ-TM who received HSCT between 1998 and 2009 at five centers in China's Mainland. Both related (n = 35) and unrelated donors (n = 15) with complete human leukocyte antigen matches were included. The stem cell sources included bone mar-row (BM), peripheral blood stem cells, umbilical cord blood (UCB) and a combination of BM and UCB or a combination of BM and peripheral blood stem cells from a single sibling donor. Results The probabilities of 5-year overall survival (OS) and thalassemia-free survival (TFS) after the first HSCT were 83.1 and 67.3%, respectively. Graft failure (GF) occurred in 17 patients. Univariate analyses showed that umbilical cord blood transplantation (UCBT) was one of the potential risk factors for decreased OS (P = 0.051), and that UCBT (P = 0.002) was potentially related to TFS. GF incidence was distinct between the UCBT and non-UCBT groups (P = 0.004). Four cases of UCB-BM combined transplantation led to decreased risks of mortality and recurrence. In the UCBT group, related donor transplantation produced more favorable results than unrelated donor transplantation in OS (P = 0.009) but not in TFS (P = 0.217). Conclusions GF was the primary cause of UCBT failure. Though UCBT from related donors was not favorable, the combined transplantation of UCB and BM could improve the prognosis of UCBT.

Comparative outcomes between cord blood transplantation and bone marrow or peripheral blood stem cell transplantation from unrelated donors in patients with hematologic malignancies： a single-institute analysis

Background Umbilical cord blood （UCB） has grown substantially as an alternative source of hematopoietic stem cells for unrelated donor transplantation in both adult and pediatric patients. Our aim was to assess the leukemia-free survival （LFS） and some primary results, such as hematologic recovery, risk of graft-versus-host disease （GVHD）, relapse, and long-term survival, after unrelated cord blood transplantation compared with the outcomes of transplantations from other unrelated graft source. Methods The clinical outcomes of 112 consecutive patients with acute leukemia who received umbilical cord blood （UCBT） as a primary unrelated stem cell source （n=38）, bone marrow （UBMT n=28, transplanted before January 2003）, or peripheral blood stem cells （UPBSCT n=46, transplanted after January 2003） between July 2000 and July 2008 were analyzed. Results Except that the patients were much younger in the UCBT group （median age, 10.5 years in UCBT, 30 years in UPBSCT, and 20 years in UBMT）, other pre-transplant parameters, such as gender, diagnosis, and the phase of disease, were comparable. All patients received myeloablative regimens, primarily including BUCY; however, there was less anti- thymocyte globulin （ATG） used for the UBMT patients （2/38 in UCBT, 0/46 in UPBSCT, and 8/28 in UBMT did not use ATG, P=0.000）. Significant delays in engraftment occurred after UCBT for both neutrophil cells and platelets. The cumulative allo-engraftment rates were also significantly lower （87.8% vs. 97.8% vs. 100% for WBC, P=0.000; 73.0% vs. 97.5% vs. 89.5% for PLT, P=0.000） for UCBT. The incidence of Grade 2-4 and 3-4 acute graft versus host disease （aGVHD） was much higher in the UBMT group but did not differ among the other groups （51% and 13.2%, 40.2% and 10.5%, and 77.4% and 41.2%, respectively, for UCBT, UPBSCT, and UBMT, P=0.000）. The occurrence of extensive chronic GVHD （cGVHD） was significantly decreased for recipients of UCBT （4%） compared with that of UPBSCT （39.1%） and UBMT （49.1%, P=0.000）, although the rates of whole cGVHD were not significantly different （30.3%, 63.1%, and 60.1% for UCBT, UPBSCT, and UBMT, respectively）. The patients had a similar rate of CMV infection （21/38, 28/46, and 22/28 for UCBT, UPBSCT, and UBMT, respectively）, while the HC occurrence was lower after UCBT （7/38, 16/46, and 14/28 for UCBT, UPBSCT, and UBMT, respectively）. As of August 2012, there was no apparent difference in 5-year overall survival （OS）, LFS, or the relapse rate for each graft source （52.5%, 52.6%, and 20.8% in UCBT; 48.7%, 46.4%, and 27.9% in UPBSCT; and 46.4%, 42.9%, and 16.0% in UBMT）. Conclusion These data support the use of UCB donors as an alternative allogeneic donor.

单倍体造血干细胞联合脐血双移植治疗儿童再生障碍性贫血的临床观察

目的:探索单倍体造血干细胞移植联合脐血输注治疗儿童再生障碍性贫血的疗效及安全性。方法:纳入河南中医药大学人民医院2021年1月1日至2023年9月15日接受脐血联合单倍体造血干细胞移植治疗的9例再生障碍性贫血患儿,中位年龄为11(2-13)岁,中位随访18(7.5-21)个月,回顾性分析其临床资料,对植入情况、GVHD发生率、感染等并发症及生存进行分析。结果:9例患儿全部成功植入。中性粒细胞和血小板植入中位时间分别为11.11±1.27 d、12.44±3.36 d。1例发生Ⅰ度急性消化道移植物抗宿主病,治疗后好转,后期该例患者发生浅表性胃炎及慢性消化道移植物抗宿主病,目前临床随访中。无一例患儿发生Ⅱ-Ⅳ度急性移植物抗宿主病。出血性膀胱炎3例,CMV感染5例,细菌及真菌感染5例,经对症治疗后好转。9例患儿移植后1个月内均表现为完全供者嵌合,随访两年,9名患儿全部生存,未出现复发或发生Ⅱ-Ⅳ度移植物抗宿主病,无移植相关死亡患儿。结论:单倍体造血干细胞移植联合脐血输注治疗儿童再生障碍性贫血时GVHD发生率低,程度轻,疗效显著,可以作为无HLA全相合配型患儿的一种治疗方案。

脐带血干细胞移植治疗异染性脑白质营养不良3例报告

目的 探讨脐带血造血干细胞移植(UCBT)治疗异染性脑白质营养不良(MLD)的安全性及有效性。方法 回顾性分析2019年4月至9月行非血缘UCBT治疗的3例MLD患儿的临床资料。结果 3例患儿均表现为走路障碍、进行性运动功能倒退,2例出现智力落后;3例均出现溶酶体芳基硫酸脂酶A(ARSA)下降,头部MRI均示脑白质不同程度的异常信号。3例均为ARSA基因复合杂合突变。3例均行非血缘UCBT,使用氟达拉滨+环磷酰胺+兔抗人胸腺细胞免疫球蛋白+白消安方案预处理,环孢素+吗替麦考酚酯+甲氨蝶呤预防移植物抗宿主病(GVHD)。3例均植入成功,移植后1个月内全血均达到完全供者嵌合状态,头部MRI均提示脑实质病变逐渐停止进展,ARSA均逐渐恢复正常。3例患儿均出现感染,2例出现急性移植物抗宿主反应(aGVHD),1例出现慢性移植物抗宿主反应(c GVHD),经抗感染及调节免疫治疗后均好转。末次随访时间为2022年8月,3例患儿均存活,其远期疗效及神经系统恢复情况仍需长期随访。结论 UCBT是阻止MLD进展的一种安全及有效治疗手段。

全程护理干预在造血干细胞移植供者行血细胞分离单采术中的应用研究

探讨全程护理干预在造血干细胞移植供者行血细胞分离单采术中的应用。选取广东省第二人民医院2019年1月-2022年12月190例造血干细胞移植供者作为研究对象。其中,实施全程护理干预前即2019年1月-2020年12月98例供者为对照组,实施全程护理干预后即2021年1月-2022年12月92例供者为观察组。比较两组供者不良反应发生率和护理工作满意度。结果显示,实施全程护理干预前后的造血干细胞移植供者血细胞分离单采均采集成功,实施全程护理干预后,观察组供者采集不良反应发生率显著低于对照组,差异具有统计学意义(P<0.05),观察组供者对护理工作的满意度明显高于对照组(P<0.05)。研究发现,于造血干细胞移植供者行血细胞分离单采术中实施全程护理干预,能有效降低供者不良反应发生率,提升供者对护理工作的满意度。