细胞因子介导的人脐血单个核细胞体外扩增并重建NOD/SCID小鼠造血及免疫功能

背景:细胞因子介导的脐血造血细胞体外扩增有望能解决脐血移植数量不足的问题。目的:实验拟确立在无基质培养条件下体外扩增脐血单个核细胞最合适的细胞因子组合及干细胞因子、FLT3配基协同促聚核细胞生成因子对造血及免疫重建功能的影响。方法:将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7d,根据不同细胞因子组合分组,将3因子干细胞因子+FLT3配基+促聚核细胞生成因子组合在无血清无基质条件下扩增培养7d前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠。在扩增培养0,7d检测脐血CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数。脐血移植6周后通过流式细胞仪,PCR法检测存活小鼠体内的人源性细胞。结果与结论:移植6周后,存活小鼠体内均可检测到人源性CD45+细胞,扩增脐血移植组的NOD/SCID小鼠存活率和人特异性基因捡出率均高于新鲜脐血移植组和高于生理盐水移植组(P0.05)。扩增脐血组存活NOD/SCID小鼠骨髓中可检测到人髓系细胞(CD33+),T淋巴细胞(CD4+),B淋巴细胞(CD19+)和人造血干细胞成分(CD34+)细胞的表达。结果提示干细胞因子+FLT3配基+促聚核细胞生成因子3因子组合脐血造血细胞体外扩增是最合适的细胞因子组合,其扩增的脐血单个核细胞能够植入并重建NOD/SCID小鼠的造血及免疫功能。     

培养前后脐血CD34~＋细胞在NOD/SCID鼠体内造血重建功能的比较

目的:以NOD/SCID小鼠为模型, 经半致死剂量照射后输注新鲜或培养后的造血细胞, 以比较培养前后脐血CD34 细胞的造血重建功能.方法:从新鲜脐血中分离单个核细胞(MNC), 采用干细胞因子(SCF)、血小板生成素(TPO)、Flt3配体(FL)、白细胞介素3(IL-3)和白细胞介素6(IL-6)细胞因子组合体外培养14 d.通过MiniMACS免疫磁性吸附柱从新鲜或培养后的MNC中分离CD34 细胞, 4×105个CD34 细胞和5×106CD34-细胞混合后通过尾静脉输注入NOD/SCID小鼠中.饲养过程中动态观察外周血象恢复情况, 6周后检测小鼠骨髓和脾脏细胞中人源细胞及各系造血细胞的含量.结果:体外培养MNC 14 d后, 总细胞扩增了1.78倍;细胞移植6周后, 输注新鲜和培养后造血细胞的小鼠均存活, 在小鼠骨髓和脾脏中均可检测到人源细胞及各系人源血细胞和人特异ALU基因序列, 小鼠外周血象恢复到辐照前水平.培养后CD34 细胞在小鼠体内的植入水平与新鲜CD34 细胞的相近, 而其各系人源血细胞的含量高于新鲜CD34 细胞. 结论:体外培养14 d后的CD34 细胞仍保持了体内植入和重建造血的能力, 且其多系造血重建能力优于新鲜CD34 细胞.     

rhTPO对巨核系的定向分化效应

目的 :探讨重组人促血小板生成素 (rhTPO)对外周造血干细胞中巨核系祖细胞的定向诱导分化能力。方法 :经化疗及G CSF动员后分离的外周造血干细胞 ,进行液体和集落培养 ,研究rhTPO单独及与IL 3、IL 6、SCF的协同作用。结果 :液体培养后 ,TPO组单个核细胞数 (MNC)扩增了 ( 1.73± 0 .49)倍 ,IL 3 IL 6 SCF组MNC比种植时增加 ( 4.2 0± 1.14)倍 ,IL 3 IL 6 SCF TPO组MNC增加了 ( 4.5 3± 1.2 7)倍。单独应用TPO及TPO与IL 3、IL 6、SCF合用产生高比例的CD41a 细胞 ,TPO组培养前CD41a 细胞为 11.70 %± 5 .2 3% ,培养后CD41a 细胞为 19.17%± 6 .2 6 % ( P <0 .0 5 )。CD41a 细胞数在TPO组扩增了 ( 3.5 2± 1.18)倍 ,IL 3 IL 6 SCF组扩增了 ( 5 .32± 1.79)倍 ,IL 3 IL 6 SCF TPO组扩增了 ( 6 .94± 2 .19)倍。结论 :TPO可以定向诱导巨核系细胞的分化 ,IL 3、IL 6、SCF可以协同TPO的作用 ,这在造血调控研究 ,体外定向扩增外周血干细胞 ,促进外周造血干细胞移植患者血小板的恢复具有应用前景     

早期作用造血因子对人脐血CD34+细胞的体个扩增作用

为了观察早期作用造血细胞因子SCF、FL、IL-3、IL-6、TPO单独及联合应用,对脐血CD34+细胞的体外扩增作用.我们用吸附单克隆抗体-磁珠分离系统富集人脐血CD34+细胞,在体外液体培养体系中加入不同的细胞因子扩增4周,每周取样计数有核细胞总数及集落形成细胞(CFC)数.结果表明:用磁性细胞分离仪富集脐血CD34+细胞纯度为80%～87%;一些细胞因子有明显的协同效应,其联合应用的扩增作用显著高于单因子作用;SCF+FL存在下,IL-3是有效扩增有核细胞总数及CFC的关键因子;细胞因子SCF+FL+IL-3和SCF+FL+IL-3+IL-6组合对有核细胞总数及CFC均有良好的扩增效应,培养2周时对CFC的扩增倍数分别为38.3±4.4 和29.6±2.7倍,可满足成人移植及基因治疗等的需要.     

多细胞因子组合对人脐血单个核细胞体外扩增及粘附分子和CXCR4表达的影响

目的：探讨不同细胞因子组合对人脐血单个核细胞体外扩增及扩增后粘附分子和CXCR4表达的影响。方法：将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7天,在0、7天检测有核细胞数（TNC）、CD34^＋细胞数及CD34^＋CXCR4^＋、CD34^＋CD49d^＋、CD34^＋CD62L^＋的细胞数和集落形成单位（CFU）数。根据不同细胞因子组合实验分组为：对照组;SCF＋FL（简称SF）组;SFT（SCF＋FL＋TPO）组;SFT6（SCF＋FL＋TPO＋IL-6）组;SFTs（SCF＋FL＋TPO＋sIL-6R）组;SFT6s（SCF＋FL＋TPO＋IL-6/sIL-6R）组。结果：和对照组相比,SF、SFT、SFT6、SFTs、SFT6s组均可有效地扩增脐血造血细胞（P〈0.05）,SFT、SFT6、SFTs、SFT6s四组扩增效果优于SF,差异有显著性（P〈0.05）,但SFT和SFT6、SFTs三组之间却无明显区别（P〉0.05）,在SFT6组合基础上加入sIL-6R后,即SFT6s组能有效地扩增脐血细胞,并优于SFT、SFT6、SFTs三组（P〈0.05）;SF、SFT、SFT6、SFTs四组细胞因子组合均可提高脐血CD34＋细胞上CD49d、CD62L和CXCR4的表达,但四组之间差异无显著性（P〉0.05）,SFT6s组可明显促进脐血CD34＋细胞上CD49、CD62L和CXCR4的表达,并优于SF、SFT、SFT6、SFTs四组,差异有显著性（P〈0.05）。结论：IL-6/sIL-6R可协同SCF、FL和TPO有效地扩增脐血细胞并能促进和归巢有关的CD49d、CD62L及CXCR4表达     

两步法从CD34+造血干细胞诱导树突状细胞

目的：研究如何从有限的造血干细胞获得大量的树突状细胞（DC），为进一步研究树突状细胞的生化特性及临床应用提供技术支持。方法：利用免疫磁珠法（MACS）富集CD34^ 细胞，先在培养体系中加入FLT3配体（FL）、血小板生成素（TPO）及干细胞因子（SCF）等细胞因子，然后对富集的细胞进行扩增，扩增后的细胞在GM－CSF和IL－4的作用下诱导7d，诱导后的细胞用流式细胞仪分析树突状细胞特征性表面标记CD1a。结果：两步法能够获得大量的DC，在第一步中用TPO／FL／SCF／IL－3／IL－6来扩增CD34^ 细胞能获得最多的DC。在相同的培养时间下（3周），两步法能扩增出274倍的DC，大大的超过了直接诱导的扩增倍数（24倍）。并且，随着培养时间的增长，DC的扩增倍数不断上升。结论：两步法能从少量的脐血CD34^ 前体细胞诱导扩增出大量的DC，为从病人动员的CD34^ 细胞诱导扩增DC用于临床提供了实验依据。     

脐血体外同时扩增T、NK及干祖细胞移植治疗BALB/c小鼠白血病

目的观察体外同时扩增T、NK及干祖细胞脐血用于移植治疗BALB/c小鼠白血病效果.方法在不同的细胞因子组合作用下定向扩增脐血干祖细胞及T、NK免疫细胞.扩增7天后,5 × 106个/只脐血细胞经尾静脉接种X射线照射的白血病小鼠,观察各组小鼠存活时间及检查存活证据及微小残留白血病细胞.结果1.细胞因子SCF,IL-3,IL-6,IL-7,IL-2组合能显著扩增脐血中的CD34 、CD3 T、NK细胞.2.尾静脉接种2×106个/只K562产生可移植性白血病BALB/c裸鼠.3.BALB/c白血病小鼠移植6周后,实验组共存活鼠12只.而未移植者在60天内死于肿瘤.4.流式细胞分析移植30天后小鼠外周血中人CD3 细胞从最低新鲜脐血移植组(0.48±0.40)%,最高SCF IL-3,6,7组中为(3.01±1.25)%.RT-PCR 检测发现,在12只扩增后移植存活6周小鼠中,检测不到bcr/abl 基因.在3只新鲜脐血移植存活6周的小鼠中,1只呈现阳性,说明移植取得了造血与免疫重建.所有脐血移植小鼠均未出现明显临床GVHD 症状.结论这种扩增后含一定水平T、NK免疫细胞的脐血能重建可移植性BALB/c 白血病小鼠的造血和免疫.     

CD34~＋造血干/祖细胞在转hLIF基因腺病毒载体饲养层细胞中的扩增及其移植SCID小鼠的实验研究

目的:建立转hLIF基因腺病毒载体的饲养层细胞,观察对CD34+造血干/祖细胞的扩增作用,并研究移植辐射损伤模型SCID小鼠的效果.方法:建立转hLIF基因腺病毒载体的饲养层细胞,并用RT-PCR法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,流式细胞术检测各组增殖效果,建立辐射损伤模型SCID小鼠,将扩增后的CD34+造血干/祖细胞经CFDA SE荧光标记后移植入SCID小鼠体内,通过RT-PCR和观察荧光标记细胞来检测小鼠内的人源细胞.结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法证实有目的基因表达,免疫磁珠法分离的CD34+造血干/祖细胞纯度可达(95.6±2.58)%,与饲养层细胞共培养后CD34+造血干/祖细胞可扩增13.2倍,表面粘附分子CXCR4和CD54表达量仍较高,移植入SCID小鼠四周后,仍可见带有荧光标记的人源细胞,RT-PCR证明人源基因Alu的存在.结论:建立的转hLIF基因腺病毒载体饲养层细胞可以有效地扩增CD34+造血干/祖细胞,延缓其分化,并且有较高的移植效率和造血活性.     

FLT3配基与血小板生成素对造血调节作用的研究进展

FLT3配基(FL)主要影响造血干/祖细胞生长,影响淋巴系及髓系细胞的增殖;抑制髓系祖细胞的凋亡;促进树突细胞生长.TPO是血小板生成的主要调节因子,不仅促进巨核系造血,也作用于早期造血干/祖细胞,使早期造血干/祖细胞扩增,维持存活及抑制凋亡.FL与TPO联合,能使脐带血干细胞维持长时期的增殖和自我更新;在具有基质细胞层HESS-5细胞的无血清培养体系中,加入TPO与FL,可使CD34+CD38-细胞增殖.FL与TPO联合,对造血干/祖细胞显著扩增,有助于脐血移植的可行性.     

FLT配基与血小板生成素对造血调节作用的研究进展

FLT3配基(FL)主要影响造血干/祖细胞生长,影响淋巴系及髓系细胞的增殖;抑制髓系祖细胞的凋亡;促进树突细胞生长.TPO是血小板生成的主要调节因子,不仅促进巨核系造血,也作用于早期造血干/祖细胞,使早期造血干/祖细胞扩增,维持存活及抑制凋亡.FL与TPO联合,能使脐带血干细胞维持长时期的增殖和自我更新;在具有基质细胞层HESS-5细胞的无血清培养体系中,加入TPO与FL,可使CD34+CD38-细胞增殖.FL与TPO联合,对造血干/祖细胞显著扩增,有助于脐血移植的可行性.     

Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells.

In umbilical cord blood (UCB) transplantation, the number of nucleated cells per kilogram is a major predictive and critical factor of hematopoietic recovery. Thus, ex vivo expansion of hematopoietic UCB progenitors could potentially accelerate engraftment. Whereas Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) are considered indispensable, the role of interleukin 3 (IL-3) is still controversial: it has been reported either to support or abrogate the reconstituting ability of stem cells. By adding IL-3 we aimed to enhance the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells. Demonstrating a positive impact of IL-3 on the proliferation of all progenitor subsets, the amplification of CD34+ UCB cells was increased 20.9-fold +/- 5.4 (mean +/- standard error) in serum-free culture with FL, SCF, TPO, and IL-3 as opposed to 9.3-fold +/- 3.2 without IL-3 after 7 days. If IL-3 was included, primitive long-term culture-initiating cells and committed colony-forming cells were expanded 16.3-fold +/- 5.5 and 18.1-fold +/- 2.4, respectively, compared to 12.6-fold +/- 5.6 and 9.1-fold +/- 2.0 without IL-3. Analysis of cultured CD34+ UCB cells in sublethally irradiated nonobese diabetic/severe combined immunodeficient mice confirmed that cultured cells had preserved their repopulating potential. After 6 weeks, all mice showed multilineage engraftment with their bone marrow containing an average of 45% human CD45+ cells of the unmanipulated sample, 43% of cells after culture in the presence of IL-3, and 27% of cells after culture without IL-3. In combination with early acting cytokines, IL-3 therefore improves the ex vivo expansion of UCB stem and progenitor cells without impairing their engraftment potential.     

Fast but durable megakaryocyte repopulation and platelet production in NOD/SCID mice transplanted with ex-vivo expanded human cord blood CD34+ cells

We have previously established a stroma-free culture with Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) that allows the maintenance and the expansion for several weeks of a cord blood (CB) CD34+ cell population capable of multilineage and long-lasting hematopoietic repopulation in non-obese diabetic/ severe combined immunodeficient (NOD/SCID) mice. In this work the kinetics of megakarocyte (Mk)-engraftment that is often poor and delayed in CB transplantation, and human platelet (HuPlt) generation in NOD/SCID mice of baseline CD34+ cells (b34+), and of CD34+ cells reisolated after a 4-week expansion with FL+SCF+TPO (4w34+) were compared. With b34+ cells Mk-engraftment was first seen at week 3 (CD41+: 0.4%); 4w34+ cells allowed a more rapid Mk-engraftment (at weeks 2 and 3 the CD41+ cells were 0.3% and 0.8%). Circulating HuPlts were first seen at weeks 2 and 1, respectively. Mk-engraftment levels of b34+ and 4w34+ cells 6-8 weeks after transplantation were similar (12 +/- 3.5 versus 15 +/- 5% CD45+; 1.3 +/- 0.5 versus 1.8 +/- 0.5% CD41+ cells). Also serial transplant experiments were performed with expanded and reselected CB cells. In secondary and tertiary recipients the Mk population was detected with bone marrow fluorescence-activated cell sorter analysis; these experiments indicate the effective long-term repopulation of expanded cells. Selected CD34+ cells after a 4-week expansion with FL+SCF+TPO are more efficient in Mk engraftment than the same number of unmanipulated cells.     

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.     

Small peptide analogue of SDF-1alpha supports survival of cord blood CD34+ cells in synergy with other cytokines and enhances their ex vivo expansion and engraftment into nonobese diabetic/severe combined immunodeficient mice

The SDF-1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF-1 peptide analogue CTCE-0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE-0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt-3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE-0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38- cells, colony-forming unit-mixed [CFU-GEMMs]), erythroid (CFU-Es), myeloid (CFU-GMs), and megakaryocytic (CD61+CD41+ cells, CFU-MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE-0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF-1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.     

不同培养体系对脐带血造血干细胞扩增的影响

目的探讨不同培养体系对造血干细胞的体外扩增及其表型的改变。方法新鲜分离人脐带血单个核细胞（MNC），免疫磁珠法分选CD34^＋造血干细胞（HSC），计数富集得到的CD34^＋细胞，平均分为3组，每组含CD34^＋细胞2．2×10^5：A组（HSC＋CK）CD34^＋细胞接种于Stemline^TMⅡ无血清培养基中．加入早期作用因子FST组合（SCF、FL和TPO，质量浓度50ng／ml的SCF、质量浓度100ng/ml的TPO和FL），并于接种0d添加质量浓度20ng／mlIL-3：B组（HSC＋MSC）CD34^＋细胞接种于含MSC feeder的培养瓶．加入Stemline^TMⅡ无血清培养基：C组（HSC＋MSC＋CK）CD34^＋细胞接种于含MSC feeder的培养瓶．加入Stemline^TMⅡ无血清培养基，加入早期作用因子FST组合首剂添加IL-3（剂量同A组）。在培养后4、7、10、14d计数有核细胞总数，流式细胞术检测扩增细胞免疫表型的改变。结果0～14d培养后MNC细胞扩增数C组（HSC＋MSC＋CK）〉A组（HSC＋CK）〉B组（HSC＋MSC），P〈0．01。3组间CD34^＋细胞比例B组（HSC＋MSC）〉C组（HSC＋MSC＋CK）〉A组（HSC＋CK），P〈0．01。CD34^＋细胞绝对数也出现了明显增加，其中C组（HSC＋MSC＋CK）增加最为明显．其次是A组（HSC＋CK）。其中A组（HSC＋CK）培养4d较0d（14．68％）CD34^＋CD38^-细胞有明显增加（62．71％，P〈0．05），C组（HSC＋MSC＋CK）CD34^＋CD38^-细胞也略有增加（23．99％）：培养7d时，A组（HSC＋CK）、C组（HSC＋MSC＋CK）CD34^＋CD38^-细胞数明显下降，分别为4．44％和1.38％，而B组（HSC＋MSC）CD34^＋CD38^-细胞上升为18．92％，与0d时比较P〈0．05，与A组（HSC＋CK）、C组（HSC＋MSC＋CK）比较P〈0．05。结论MSC和细胞因子的联合应用，一方面使得总MNC细胞得到大量扩增，同时还使扩增后细胞保持CD34^＋免疫表型，培养体系中加入MSC能更有效／特异地扩增CD34^＋造虹干细胞群.     

Functional characterization of TPO-expanded CD34+ cord blood cells identifies CD34- CD61- cells as platelet-producing cells early after transplantation in NOD/SCID mice and rCD34+ cells as CAFC colony-forming cells

Transplantation of thrombopoietin (TPO)-expanded cord blood CD34(+) cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO-expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34(-) /CD61(-)/lineage(-) cells (Lin(-)), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin(-) dose dependence. The Lin(-) population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin(-) population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34(+) cells remaining after expansion (rCD34(+)) was similar to mice transplanted with nonexpanded CD34(+) cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area-forming cell colony-forming cells resided mostly in the rCD34(+) population. The pro-MK CD61(+) cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application.     

Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.     

转JAK2基因脐血CD34+细胞体外扩增与生物学特性研究

目的： 探讨转基因JAK2介导的脐血干祖细胞长期扩增调控的可行性和转基因细胞的生物学特征。方法: 构建逆转录病毒载体MGI-F₂JAK2，内含有JAK2基因的功能催化区和2个与小分子靶向基因合成药物(AP20187)结合的位点蛋白(F36v，F₂)。应用MiniMACS磁珠分选系统纯化分离脐血CD34⁺细胞，用含JAK2的逆转录病毒上清转染脐血CD34⁺细胞。转染后的CD34⁺细胞在IMDM培养体系中，将细胞分为AP20187组;FL组；TPO组；AP20187+FL+TPO (AFT) 组。对扩增后的细胞定期检测基因转移后GFP动态变化、细胞免疫标记、造血祖细胞集落培养、染色体核型分析和裸鼠致瘤实验。结果: 分选的CD34⁺细胞纯度>91%，基因转移率为49.32%±6.21%；只有AP20187+FL+TPO组可以使转基因的脐血CD34⁺细胞大量增殖，扩增至第8周时细胞数达10⁹，CD34⁺细胞GFP的阳性率由基线水平逐渐上升并于第8周时达到90%以上；细胞表型为CD33⁺、CD61⁺、Gly-A⁺部分阳性；CD38⁺、HLA-DR⁺强阳性；CD2、CD7、CD19接近阴性。扩增的CD34+细胞可分别形成BFU-E、CFU-GM、CFU-Mix并以CFU-GM集落为主。扩增后CD34⁺细胞检测染色体核型正常，裸鼠实验无致瘤特性。结论: 转染JAK2 基因的人脐血CD34⁺细胞协同FL和TPO细胞因子可以体外长期扩增脐血干祖细胞，对今后研究细胞信号转导、造血调控以及开展干细胞和基因治疗都有潜在的应用价值。     

FL、rIL—6、rIL—6R对脐血CD34^＋细胞的体外分化增殖作用

目的：探讨FL、rIL-6、rIL-6R对CD34^ 细胞的体外分化增殖作用。方法：用磁性细胞分离仪富集人脐血CD34^ 增胞。加入FL、rIL-6、rIL-6R及其不同组合因子,不同时间取样计数CD34^ 细胞集落数及细胞数。结果：FL、rIL-6、rIL-6R扩增细胞形成集落的主要成分为CFU－GM,但在rIL-6 rIL-6R及FL＋rIL-6 rIL-6R作用下,也可形成一定数量的BFU－E及CFU－M,而CFU－MK则未见形成。FL＋rIL-6 rIL-6R可刺激红细胞生成。可使CD34^ 细胞总数在第7天时扩增6．4倍。结论：FL＋rIL-6 rIL-6R可扩增脐血CD34^ 细胞数量并可促进其分化。