HUMAN PRIMITIVE HEMATOPOIETIC PROGENITOR CELLS ARE MORE ENRICHED IN CD34^+CD38^- POPULATION THAN IN CD34^+CD38^+ POPULATION

To clarify the hematopoietic potential of various sub-classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with anti-CD34 and -CD38 McAb. We characterized two cell fractions in CD34+cells with or without CD38 expression. A clonogenic assay showed that most CFC were present in CD34+CD38+ population. Morphologic analysis showed that blast-like cells were more enriched in the CD34+CD38 fraction. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell function by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At the first two weeks, more CF.C harvested from the culture in the fractions initiated with both populations. However, more LTC-IC were present during weeks 4 to 12 in the CD34+CD38- population. These results indicate the primitive progenitors are more enriched in CD34+CD38 population than in CD34+CD38+ cells.

Hematopoietic stem cells in research and clinical applications:The“CD34 issue”

In this paper,experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data.Although not clearly apparent,the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells.One of the "f irst rate" parameters in clinical transplantations in hematology;i.e.the CD34+ positive cell dose,has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34.This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors.This proportion could vary with respect to the source,pathology,treatment,processing procedure,the graft ex vivo treatment and so on.Therefore,a universal dose of CD34+ cells cannot be def ined.In addition,to avoid further confusion,the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.

Exploring hematopoietic stem cell population in human milk and its benefits for infants:A scoping review

Objective:To comprehensively explore hematopoietic stem cells(HSCs)in human milk,understanding their molecular markers,isolation methods,benefits for infants,and potential medical applications.Methods:We conducted a scoping literature review following the PRISMA-ScR guidelines.This review included studies investigating HSCs in human milk,utilizing molecular markers such as CD34^(+),CD113^(+),and CD117^(+)for characterization.Both in vitro and in vivo studies exploring the morphology,function,and clinical implications of these cells were considered.The diverse range of papers reviewed were indexed in PubMed,Science Direct,Scopus,Sage Journals,and Google Scholar,published between 2010 and 2023.Results:This scoping review explored 577 articles and selected 13 studies based on our inclusion criteria,focusing on HSCs in human milk.Most studies dilute samples prior to HSC isolation,followed by detection using markers such as CD34^(+),CD113^(+),and CD117^(+),with flow cytometry serving as the primary analysis tool,focusing on their isolation and detection methods.While no definitive benefits have been conclusively established,there is a strong belief in the potential of HSCs to positively impact infant immunity,growth,and tissue repair.Conclusions:This review presents significant evidence supporting the presence of HSCs in human milk,identified by markers such as CD34^(+),CD113^(+),and CD117^(+).These cells show considerable potential in enhancing infant health,including immunity,tissue repair,cognitive development,and gastrointestinal health.Despite methodological variations in isolation and detection techniques,the collective findings underscore the potential clinical relevance of HSCs in human milk.Moreover,this review highlights the noninvasive accessibility of human milk as a source of HSCs and emphasizes the need for further research to unlock their therapeutic potential.

Impact of T cells on hematopoietic stem and progenitor cell function:Good guys or bad guys?

When hematopoietic stem and progenitor cells(HSPC)are harvested for transplantation, either from the bone marrow or from mobilized blood, the graft contains a significant number of T cells. It is these T cells that are the major drivers of graft-vs-host disease(Gv HD). The risk for Gv HD can simply be reduced by the removal of these T cells from the graft. However, this is not always desirable, as this procedure also decreases the engraftment of the transplanted HSPCs and, if applicable, a graft-vs-tumor effect. This poses an important conundrum in the field: T cells act as a double-edged sword upon allogeneic HSPC transplantation, as they support engraftment of HSPCs and provide anti-tumor activity, but can also cause Gv HD. It has recently been suggested that T cells also enhance the engraftment of autologous HSPCs, thus supporting the notion that T cells and HSPCs have an important functional interaction that is highly beneficial, in particular during transplantation. The underlying reason on why and how T cells contribute to HSPC engraftment is still poorly understood. Therefore, we evaluate in this review the studies that have examined the role of T cells during HSPC transplantation and the possible mechanisms involved in their supporting function. Understanding the underlying cellular and molecular mechanisms can provide new insight into improving HSPC engraftment and thus lower the number of HSPCs required during transplantation. Moreover, it could provide new avenues to limit the development of severe Gv HD, thus making HSPC transplantations more efficient and ultimately safer.

人造血干/祖细胞多态性的研究:Ⅲ.骨髓CD34^+造血干/祖细胞功能亚群的分析

CD 34^+造血干/祖细胞(HSC/HPC)是十分异质性的,由多个不同功能亚群所构成,在分化方向与重建造血等方面差异显著。本文对正常人骨髓CD 34^+ HSC/HPC各亚群进行了较全面的分析。首先以阳性选择策略,采用Isolex^(TM) 50免疫磁球分选术富集骨髓CD 34^+HSC/HPC,其纯度>90%,随之采用免疫荧光抗体双标记二维流式细胞仪对其测定,发现高纯度的CD 34^+HSC/HPC可分为八个不同亚群:1.CD 34^+/CD 71^-(23.4%—56.6%)与CD 34^+/CD 71^+(33.4%—66.6%);2.CD34^+/CD 45^-(80.8%—82.5%)与CD34^+/CD 45^+(8.1%—11.2%);3.CD 34^+/CD 33^-(20.4%—80.6%)与CD 34^+/CD 33^+(14.6%—64.8%);4.CD 34^+/DR^-(6.3%—11.0%)与CD 34^+/DR^+(82.8%—85.5%)。用免疫胶体金——免疫桥酶联组化双染色对上述亚群进一步分析的结果与流式细胞仪的高度一致。

CD_(34)^+造血干细胞混合CD_3^+ T细胞移植分离移植物抗宿主病和移植物抗白血病效应

目的 探讨在异基因造血干细胞移植中能否采用纯化 CD34 + 造血干细胞混合一定量纯化 CD3+ T细胞移植达到既控制移植物抗宿主病 (GVHD)又保留移植物的抗白血病效应 (GVL )。 方法 (C5 7BL /6× 6 15 ) F1代接种 L 6 15白血病细胞株建立小鼠白血病模型作为移植受鼠 ,利用免疫磁珠纯化骨髓 CD34 + 造血干细胞及脾脏CD3+细胞 ,流式细胞仪分析纯化前后 CD34 +、CD3+细胞百分比 ,观察单纯移植 CD34 +细胞及混合不同数量级 CD3+细胞后受鼠生存时间、GVHD、GVL效应 ,移植后骨髓造血重建细胞来源。 结果 纯化 CD34 + 细胞 1× 10 6 移植组10 0 %死于白血病复发 ;CD34 +细胞 +纯化 CD3+细胞 (1× 10 7)移植组生存期明显延长 ,5 0 %死于白血病复发 ,无GVHD表现 ;CD34 + 细胞 +CD3+ 细胞 (5× 10 7)移植组长期存活 ,无白血病复发 ,无 GVHD表现 ;CD34 + 细胞 +CD3+细胞 (1× 10 8)移植组 6 2 .5 %死于 GVHD,37.5 %长期存活 ;CD34 + 细胞 +CD3+ 细胞 (1.5× 10 8)移植组 10 0 %死于GVHD。移植后生存时间 >6 0 d的受鼠骨髓造血重建细胞 y染色体检出率为 10 0 %。 结论 移植物中 CD3+细胞数量与 GVHD和 GVL效应密切相关 ,通过限定移植物中的 CD3+ 淋巴细胞的含量能够保留

Apoptotic bone marrow CD34+ cells in cirrhotic patients

AIM: To access the frequency and level of apoptotic CD34+ cells isolated from the marrow fluid of patients with post-hepatitis cirrhosis. METHODS: The frequency of bone marrow CD34+ cells and apoptotic bone marrow CD34+ cells in 31 inpatients with post-hepatitis cirrhosis (cirrhosis group), and 15 out-patients without liver or blood disorders (control group) was calculated by flow cytometry. Parameters were collected to evaluate liver functions of patients in cirrhosis group. RESULTS: The percentage of normal bone marrow CD34+ cells was 6.30% ± 2.48% and 1.87% ± 0.53% (t = 3.906, P < 0.01) while that of apoptotic marrowCD34+ cells was 15.00% ± 15.81% and 5.73% ± 1.57% (t = 2.367, P < 0.05) in cirrhosis and control groups, respectively. The percentage of apoptotic marrow CD34+ cells was 6.25% ± 3.30% and 20.92 ± 18.5% (t = 2.409, P < 0.05) in Child-Pugh A and Child-Pugh B + C cirrhotic patients, respectively. The percentage of late apoptotic marrow CD34+ cells was positively correlated with the total bilirubin and aspartate aminotransferase serum levels in patients with cirrhosis. CONCLUSION: The status of CD34+ marrow cells in cirrhotic patients may suggest that the ability of hematopoietic progenitor cells to transform into mature blood cells is impaired.

Effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD_(34)^+ hematopoietic cells in bone marrow of asthmatic mice

BACKGROUND: Corticosteroids remain the most effective therapy available for asthma. They have widespread effects on asthmatic airway inflammation. However, little is known about the effects of corticosteroids on the production of bone marrow inflammatory cells in asthma. This study observed the effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD34+ hematopoietic cells, so as to explore the possible effectiveness of a bone marrow-targeted anti-inflammatory strategy. METHODS: Balb/c mice were sensitized and challenged with ovalbumin (OVA) to establish an asthmatic model. For two consecutive weeks, asthmatic mice were challenged with OVA while being given either prednisone, montelukast, prednisone plus montelukast, or sterile saline solution. The mice were killed 24 hours after the last challenge with OVA, and bronchoalveolar lavage fluid (BALF), peripheral blood, and bone marrow were collected. Eosinophils in peripheral blood and BALF, and nucleated cells in BALF, peripheral blood, and bone marrow were counted. The percentages of CD34+ cells, CD4+ T lymphocytes and CD8+ T lymphocytes among nucleated cells in peripheral blood and bone marrow were counted by flow cytometry. Immunocytochemistry and in situ hybridization were employed to detect expression of CD34 and interleukin (IL)-5Ralpha mRNA (CD34+ IL-5Ralpha mRNA+ cells) among bone marrow hematopoietic cells. RESULTS: Compared with the sterile saline solution group, the number of eosinophils in BALF and peripheral blood, CD34+ cells in peripheral blood and bone marrow, and CD34+ IL-5Ralpha mRNA+ cells in bone marrow of mice from the prednisone and prednisone plus montelukast groups were significantly lower (P

Higher proportions of peripheral CD19^＋CD5^＋ B cells predict the effect of corticosteroid in patients with late-onset hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation

Background The cause of late-onset hemorrhagic cystitis （LOHC） after allogeneic hematopoietic stem cell transplantation （allo-HSCT） remains obscure. In clinical practice, some LOHC patients respond to immunosuppression.The aim of this study was to determine the immune pathogenesis of LOHC post allo-HSCT.Methods With the diagnosis of LOHC, patients were given initial treatment consisting of fluid hydration, alkalization and forced diuresis, and empirical anti-viral therapy for 10-14 days or until a week after the virus became negative. The nonresponders were applied corticosteroid. Seven to ten days later, patients＇ response was evaluated. Along with treatment, CD19^＋ B lymphocyte subsets were measured at various study points.Results From October 2009 to March 2010, we found 28 cases of LOHC occurred in 25 patients who underwent allo-HSCT in our hospital. Except that three cases were not treated according to the protocol, the other 25 cases were divided into three groups： anti-virus responders （Group A, n=6）, corticosteroid responders （Group B1, n=16）,corticosteroid and anti-virus nonresponders （Group C, n=3） according to their clinical response. Percentages of CD19^＋CD5^＋ B lymphocytes were not significantly different among three groups at onset of LOCH. However, in Group B1after the first anti-virus phase, percentages of CD19^＋CD5^＋ lymphocytes significantly increased comparing with those at onset （P=0.022）, and then significantly decreased at PR （P=0.003） and CR （P=0.002） with corticosteroid treatment. But significant change was not observed in Groups A and C.Conclusion The immune etiology seems to be involved in the development of LOHC and the proportion of CD19^＋CD5^＋lymphocytes may serve as a cellular biomarker to predict the response to corticosteroid in LOHC

Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model

AIM:To investigate the effect of human umbilical cord stem cells,both mesenchymal and hematopoietic(CD34+),in the treatment of arthritis.METHODS:Mesenchymal stem cells(MSCs) and hematopoietic(CD34+) stem cells(HSC) were isolated from human umbilical cord blood obtained from the umbilical cord of healthy pregnant donors undergoing fullterm normal vaginal delivery.MSC,HSC,methotrexate(MTX) and sterile saline were injected intra-articularly into the rat hindpaw with complete freunds adjuvant(CFA) induced arthritis after the onset of disease(day 34),when arthritis had become well established(arthritis score ≥ 2).Arthritic indices were evaluated and the levels of interleukin(IL)-1,tumor necrosis factor(TNF)-α and interferon(IFN)-γ and anti-inflammatory cytokine IL-10 in serum were determined using enzyme-linked immunosorbent assay.Animals of all groups were sacrificed 34 d after beginning treatment,except positive control(PC) which was sacrificed at 10,21 and 34 d for microscopic observation of disease progression.We used hematoxylin,eosin and Masson's trichrome stains for histopathological examination of cartilage and synovium.RESULTS:The mean arthritis scores were similar in all groups at 12 and 34 d post immunization,with no statistical significant difference.Upon the injection of stem cells(hematopoietic and mesenchymal),the overall arthritis signs were significantly improved around 21 d after receiving the injection and totally disappeared at day 34 post treatment in MSC group.Mean hindpaw diameter(mm) in the MSC rats was about half that of the PC and MTX groups(P = 0.007 and P = 0.021,respectively) and 0.6 mm less than the HSC group(P = 0.047),as indicated by paw swelling.Associated with these findings,serum levels of TNF-α,IFN-γ and IL-1 decreased significantly in HSC and MSC groups compared to PC and MTX groups(P < 0.05),while the expression of IL-10 was increased.Histopathological examination with H and E stain revealed that the MTX treated group showed significant reduction of leucocytic infiltrate and hypertrophy of the synovial tissue with moderate obliteration of the joint cavity.Stem cells treated groups(both hematopoietic CD34+ and mesenchymal),showed significant reduction in leucocytic infiltrate and hypertrophy of the synovial tissue with mild obliteration of the joint cavity.With Masson's trichrome,stain sections from the PC group showed evidence of vascular edema of almost all vessels within the synovium in nearly all arthritic rats.Vacuoles were also visible in the outer vessel wall.The vessel became hemorrhagic and finally necrotic.In addition,there was extensive fibrosis completely obliterating the joint cavity.The mean color area percentage of collagen in this group was 0.324 ± 0.096,which was significantly increased when compared to the negative control group.The mean color area percentage of collagen in hematopoietic CD34+ and mesenchymal groups was 0.176 ± 0.0137 and 0.174 ± 0.0197 respectively,which showed a marked decrement compared to the PC group,denoting a mild increase in synovial tissue collagen fibers.CONCLUSION:MSC enhance the efficacy of CFAinduced arthritis treatment,most likely through the modulation of the expression of cytokines and amelioration of pathological changes in joints.

Preferential expression of cytochrome CYP CYP2R1 but not CYP1B1 in human cord blood hematopoietic stem and progenitor cells

Cytochrome P450(CYP)enzymes metabolize numerous endogenous substrates,such as retinoids,androgens,estrogens and vitamin D,that can modulate important cellular processes,including proliferation,differentiation and apoptosis.The aim of this study is to characterize the expression of CYP genes in CD34+human cord blood hematopoietic stem and early progenitor cells(CBHSPCs)as a first step toward assessment of the potential biological functions of CYP enzymes in regulating the expansion or differentiation of these cells.CD34+CBHSPCs were purified from umbilical cord blood via antibody affinity chromatography.Purity of CD34+CBHSPCs was assessed using fluorescence-activated cell sorting.RNA was isolated from purified CD34+CBHSPCs and total mononuclear cells(MNCs)for RNA-PCR analysis of CYP expression.Fourteen human CYPs were detected in the initial screening with qualitative RT-PCR in CD34+CBHSPCs.Further quantitative RNA-PCR analysis of the detected CYP transcripts yielded evidence for preferential expression of CYP2R1 in CD34+CBHSPCs relative to MNCs;and for greater expression of CYP1B1 in MNCs relative to CD34+CBHSPCs.These findings provide the basis for further studies on possible functions of CYP2R1 and CYP1B1 in CBHSPCs'proliferation and/or differentiation and their potential utility as targets for drugs designed to modulate CD34+CBHSPC expansion or differentiation.

Characteristics of Ex Vivo Expansion of Endothelial Progenitor Cells

Summary： The characteristics for the ex vivo expansion of the endothelial progenitor cells （EPCs） were explored, CD34^＋ cells were selected from umbilical cord blood mononuclear cells （MNC） by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34^＋ and CD34 from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor （VEGF） and passage on cell differentiation, expansion kinetics and apoptosis were examined, EPCs were determined and quantified by immunocytochemistry and flow cytometry, The results showed that both coculture of CD346＋ and CD34^- and total MNC led to a significant increase in the expansion of CD34^＋ cells as compared with CD34 enrichment （P〈0.05）. There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis （P〉0.05）, These differentiated EPCs were positive for CD34^＋, von Willebrand factor （vWF）, KDR, CD31 staining and phagocytized acetylated low-density lipoprotein （LDL）. CD34^＋ cells accounted for （68.2±6,3） % of attaching （AT） cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34^＋ and CD34^- or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.

CD34^＋造血祖细胞的定向诱导分化研究

加强造血调控的基础与临床研究裴雪涛血细胞的生成及其调控涉及生命科学中的许多热点问题，如细胞的增殖、分化、迁移、发育、成熟、衰老、凋亡、癌变，也涉及细胞因子及其受体的相互作用与信号传导，细胞与细胞、细胞与基质的相互作用等。同时，许多血液恶性肿瘤、造血障...

人正常骨髓 CD_(34)^+ 造血细胞的形态学与细胞化学特征

目的：探讨ＣＤ３４＋造血细胞的形态学特征。方法：采用免疫磁珠－流式细胞仪分选二步法获取高纯度的人正常骨髓ＣＤ３４＋造血细胞。结果和结论：ＣＤ３４＋造血细胞从形态上可分为３型，Ⅰ型胞体略大于淋巴细胞，各种细胞化学染色阴性，最原始，拟定候选干细胞群；Ⅱ型为典型的小淋巴细胞状，细胞化学亦为阴性，拟定多向祖细胞阶段；Ⅲ型胞体极不均一，依分化方向不同其细胞化学表现为±～＋＋，拟定定向祖细胞阶段。

Lin^-CD_(34)^-与Lin^-CD_(34)^+细胞差异表达基因的鉴定

目的 克隆并鉴定Lin-CD3 4-细胞区别于Lin-CD3 4+细胞的可能与其性状相关的基因。方法 以Lin-CD3 4-细胞作为检测对象 ,Lin-CD3 4+细胞作为驱动细胞 ,应用抑制消减杂交技术构建Lin-CD3 4-细胞的cDNA消减文库。选取部分克隆测序 ,测序结果与GenBank进行同源性比较和功能分析。结果 共获得 593个含有插入片段的克隆 ,片段的长度为 30 0～ 50 0bp。随机选取 53条EST进行测序 ,其中 37条EST代表了 1 0个已知基因 ,其余 1 6条EST代表了 4个未知序列。结论 利用抑制消减杂交技术鉴定出部分Lin-CD3 4-细胞特异表达的基因 ,可能与Lin-CD3

体外扩增脐血CD_(34)^+细胞

目的探讨某些造血细胞因子对脐血造血干细胞的扩增效应。方法脐血ＣＤ＋３４细胞经免疫磁珠法分选后，在含有重组人Ｆｌｔ３配基（ｒｈＦｌｔ３Ｌ）、干细胞因子（ｒｈＳＣＦ）、白细胞介素１β（ｒｈＩＬ１β）、ｒｈＩＬ３和ｒｈＩＬ６的液体培养体系中培养７至１４天。采用流式细胞仪、造血细胞集落和长期培养起始细胞（ＬＴＣＩＣ）方法检测扩增后的细胞。结果在上述细胞因子作用下，脐血ＣＤ＋３４细胞明显扩增。细胞总数、ＣＤ＋３４细胞和造血细胞集落总数在第７天分别扩增３１±１９、４±５、１０±６７倍、第９天分别扩增２３６±１０６、２０±２０、５７±６２，第１４天分别扩增７７０±５０５、３０±３０、１８９±１３３。扩增的细胞以髓系祖细胞为主，极少红系祖细胞，无淋巴系细胞，并含有ＬＴＣＩＣ。结论理想的造血细胞因子的组合可以加速体外扩增脐血ＣＤ＋３４细胞。

选择性CD_(34)^+细胞和非选择性自体外周血干细胞移植治疗多发性骨髓瘤的比较研究

目的 明确选择性CD3 4+ 细胞自体外周血干细胞移植 (APBSCT)治疗多发性骨髓瘤 (MM)的临床效果。方法 对 2 1例接受CD3 4+ 细胞APBSCT治疗的MM患者 (选择组 )和另外 2 1例接受非选择性APBSCT的MM患者 (对照组 )的造血恢复时间、有效率、生存率、移植相关不良反应、移植费用进行比较。两组病例在年龄、初诊时血清 β2 微球蛋白水平和移植时疾病状态均具有可比性 ,诱导治疗及预处理方案基本相同。结果 与对照组相比 ,选择组患者回输的CD3 4+ 细胞数显著偏低 [对照组和选择组分别为 9.4(1.1～ 15 .0 )× 10 6/kg和 2 .2 (0 .5～ 14.3)× 10 6/kg](P <0 .0 0 1) ,中性粒细胞恢复至≥ 0 .5× 10 9/L和血小板恢复至≥ 2 0× 10 9/L的中位时间 ,在选择组分别为 10d和 9d ,对照组分别为 9.5d(P=0 .35 7)和 4.5d(P =0 .0 0 5 )。两组的治疗有效率相似 (选择组 85 .7% ,对照组 90 .4% ) ,3年无病生存率 (选择组和对照组分别为 32 %和 39% )和总生存率 (选择组和对照组分别为 85 %和 79% )两组相比差异无显著性 ,而且使用非选择性APBSCT可明显降低移植费用。结论 选择性CD3 4+ 细胞APBSCT与非选择性APBSCT相比 ,并不能改善MM的临床治疗结果 ,而且移植费用更大。

A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34^+ cells to improve combination chemotherapy tolerance

OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.

CliniMACS系统进行CD^＋34造血干细胞阳性富集的应用评价

目的探讨CliniMACS系统进行CD^＋34造血干细胞阳性富集的有效性、安全性及应用效果。方法对39例实体瘤和自身免疫性疾病患者动员后采集的外周血干细胞进行了CliniMACS系统阳性富集的CD^＋34自体造血干细胞移植。结果阳性富集后CD^＋34细胞纯度平均可达89．8％，CD^＋34细胞平均回收率达78．9％，粒细胞-巨噬细胞集落形成单位（CFU—GM）数量为（15．0±7．0）×10^5／kg，阳性富集前、后细胞胎盘蓝拒染率比较差异无统计学意义，保持了细胞良好的生物学活性，移植后于2～3周内造血重建。结论应用CliniMACS系统进行CD^＋34造血干细胞阳性富集，可安全、有效地应用于造血干细胞移植。