不同动员方案对异基因造血干细胞移植临床疗效的影响

目的 探讨异基因造血干细胞移植中不同动员方案的临床效果.方法 回顾性分析71例异基因外周血造血干细胞移植的临床资料,根据供者采用动员剂的不同分为G-CSF动员组(G组,有24例受者)和G-CSF联合GM-CSF动员组(G+M组,有47例受者).比较两组供者的动员效果及移植物细胞成分,观察受者术后造血功能重建的情况和GVHD的发生情况,观察供者应用动员剂后的不良反应.结果 动员4 d后,G组供者的外周血白细胞计数为(49.6±19.5)×109/L,明显高于G+M组供者的(25.4±10.4)×109/L(P＜0.05).两组间CD34+细胞占单个核细胞比例的差异无统计学意义(P＞0.05),但G+M组CD34+CD38-细胞占CD34+细胞的比例为(37.7±5.7)%,明显高于G组的(31.4±4.5)%(P＜0.05).两组供者经过1～3次采集均能获取足够的CD34+细胞,两组采集的供者淋巴细胞计数及其亚群分布的差异均无统计学意义(P＞0.05).两组受者间CD34+细胞、CD34+CD38-细胞及T淋巴细胞亚群输入量的差异均无统计学意义(P＞0.05).术后所有受者的造血功能均顺利重建.术后对受者进行2～55个月的随访,无论是急性还是慢性GVHD,其发病率和严重程度在两组间的差异均无统计学意义(P＞0.05).术后共有17例受者死于原发病复发,10例死于GVHD和感染等移植相关并发症,G组和G+M组分别有14例(58.3%)和31例(66.0%)受者存活.在使用动员剂后,供者出现的主要不良反应为骨骼肌酸痛和发热,多发生在用药后36 h,给予解热镇痛药后缓解.结论 单用G-CSF与联合应用G-CSF和M-CSF进行动员的临床效果相当,但后者对CD34+CD38-细胞的选择性较强,而在异基因造血干细胞移植输入较多的CD34+细胞和CD34+CD38-细胞有利于受者造血功能的快速重建.     

异基因小鼠髓腔内骨髓移植诱导免疫耐受

目的探讨异基因小鼠髓腔内骨髓移植(IBM-BMT)诱导免疫耐受的效果。方法雄性BALB/c小鼠和雌性C57BL/6小鼠分别作为骨髓移植的供、受者。受者预处理后进行IBM-BMT,建立异基因小鼠骨髓移植模型。通过皮肤移植和混合淋巴细胞反应(MLR)对受者的耐受状态进行检测。结果接受过IBM-BMT的受者进行供者来源的皮肤移植,移植物的存活时间>300d,较对照组的(12.7±1.63)d明显延长(P<0.01),而受者接受来自无关供者(KM小鼠)的皮肤移植物存活时间未见延长。接受过IBM-BMT的受者脾细胞对供者脾细胞的MLR增殖率均明显降低,与对照组比较,P<0.01,而对无关供者的脾细胞仍表现强烈的增殖反应。结论应用IBM-BMT可以诱导受者获得供者抗原的特异性免疫耐受,使移植物存活时间延长。     

供者使用粒系集落刺激因子后供髓对异基因骨髓移植受者的影响

目的　观察供者使用粒系集落刺激因子 (G CSF)后供髓对异基因骨髓移植受者造血重建及移植物抗宿主病 (GVHD)发生的影响。方法　用HLA相合 ,混合淋巴细胞培养 (MLC)阴性的供者骨髓 ,给受者行异基因骨髓移植 ,在预处理方案、GVHD的预防及支持治疗方法相同的条件下 ,比较研究组 30例供者应用G CSF 3～ 4μg·kg-1·d-1,连用 7d后采髓和对照组 15例供者常规采髓对受者异基因骨髓移植造血重建和GVHD发生的作用。结果　在类同采髓量中研究组所获单核细胞(MNC)、粒细胞巨噬细胞集落生成单位 (CFU GM )及CD34数显著大于对照组 (P <0 .0 1)。研究组受者移植后中性粒细胞计数 >0 .5× 10 9/L与血小板 >2 0× 10 9/L时间分别为 (16 .7± 3.2 )d与 (18.4± 3.0 )d ,对照组为 (2 2 .5± 5 .1)d与 (2 6 .3± 5 .90 )d ,P <0 .0 1,研究组造血重建加速。研究组受者急性Ⅱ Ⅳ度GVHD发生 1例 (发生率 3 .3% ) ,对照组发生率 2 6 .7% (P <0 .0 5 )。慢性GVHD两组差异无显著性 (P >0 .0 5 )。输入的T淋巴细胞总数无明显改变 ,通过比较 ,研究组受者CD4减少 ,CD8增加 (P <0 .0 1)。结论　HLA相合的异基因骨髓移植 ,使用经G CSF动员的供者骨髓 ,可使受者造血重建加快 ,尤其减轻重度急性GVHD ,这一作用可能与骨髓内T淋巴细胞亚群     

基因工程Treg细胞对小鼠异基因骨髓移植后GVHD和GVL效应的影响

目的 探讨输注慢病毒载体介导的小鼠基因工程调节性T淋巴细胞(Treg细胞)对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)及移植物抗白血病(GVL)效应的影响.方法 利用慢病毒载体介导,将小鼠叉状头螺旋转录因子(Foxp3)基因转导入Balb/c小鼠的CD4+CD25-T淋巴细胞,即为基因工程Treg细胞.以Balb/c小鼠为供者.C57BL/6小鼠为受者,进行异基因骨髓移植,移植当天受者接受X线直线加速器全身照射.用随机数字表法将受者分为5组,每组10只.(1)单纯照射组:经受者尾静脉输注RPMI 1640培养液0.2 ml;(2)白血病对照组:经受者尾静脉输注供者骨髓细胞5×106个+C57BL/6小鼠T淋巴细胞白血病/淋巴瘤细胞株(EL4细胞)500个;(3)移植对照组:经受者尾静脉输注供者骨髓细胞5×106个+脾细胞5×106个+EL4细胞500个;(4)工程Treg组:经受者尾静脉输注供者骨髓细胞5×106个+脾细胞5×106个+EL4细胞500个+基因工程Treg细胞5×106个;(5)空载体对照组:经受者尾静脉输注供者骨髓细胞5×106个+脾细胞5×106个+EL4细胞500个+空载体转导的CD4+CD25-T淋巴细胞5×106个.每天观察受者存活情况;记录GVHD及白血病的发生情况;各组均于小鼠濒死前取其肝脏、小肠、皮肤、脾脏等组织,进行病理学观察;取长期存活(超过60 d)受者的骨髓细胞,检测嵌合情况.结果 单纯照射组、白血病对照组、移植对照组、工程Treg组和空载体对照组小鼠存活时间分别为(10.3±1.5)d、(20.7±1.9)d、(26.0±4.3)d、(49.0±17.7)d和(24.4±4.1)d,工程Treg组小鼠存活时间明显长于其他各组,差异有统计学意义(P＜0.05).白血病对照组小鼠肝、脾组织病理切片均存在白血病细胞浸润表现,移植对照组及空载体对照组小鼠肝脏、皮肤和小肠病理切片存在GVHD病理改变,而工程Treg组长期存活小鼠各组织病理切片结构基本正常,未见GVHD及白血病细胞浸润病理表现,该组GVHD评分明显低于移植对照组及空载体对照组.结论 小鼠异基因骨髓移植时联合输注基因工程Treg细胞可有效减少GVHD的发生并保留GVL效应.     

粒细胞集落刺激因子减轻小鼠混合骨髓移植后移植物抗宿主病的实验研究

目的探讨同基因骨髓混合一定比例粒细胞集落刺激因子(G-CSF)动员的异基因骨髓移植能否减轻急性移植物抗宿主病(aGVHD).方法将BALB/c与BCF1(BALB/c×C57BL/6)小鼠或与G-CSF动员BCF1小鼠脾细胞按一定比例混合,腹腔注入BALB/c幼鼠,制备新生小鼠GVHD模型,结果以脾指数表示.成年雌性BALB/c小鼠接受60Co全身照射8.5Gy后进行移植,移植物为BALB/c与雄性BCF1或与G-CSF动员BCF.小鼠骨髓细胞按一定比例的混合,移植细胞总数60×105个/只.观察移植小鼠aGVHD典型症状、病理表现及存活率.ELISA法测定细胞因子含量,流式细胞术分析T细胞亚群变化.结果(1)注射BALB/c与BCF1小鼠脾细胞混合比例为21、11及异基因BCF1小鼠脾细胞的新生小鼠均发生GVHD;但G-CSF动员与否,GVHD发生程度差异有统计学意义.(2)21及11混合骨髓移植(MBMT)组小鼠有中到重度GVHD表现;经G-CSF动员的MBMT组小鼠8周存活率较未动员组明显提高(P＜0.05).(3)G-CSF动员供鼠后L3T4+细胞下降显著,L3T4+/Lyt2+比值明显低于未动员组(P＜0.01).(4)G-CSF动员供鼠后混合淋巴细胞反应(MLR)细胞培养上清中,IL-2、IFN-γ水平降低,IL-4水平升高.结论同基因骨髓混合一定量H-2半相合异基因骨髓移植可减轻GVHD的发生;G-CSF动员供鼠可进一步减轻MBMT后GVHD的发生.其机理可能与IL-2、IFN-γ下降、IL-4升高有关.     

输注鼠基因工程Treg细胞抑制小鼠异基因骨髓移植后移植物抗宿主病

目的 探讨输注慢病毒载体介导的鼠基因工程调节性T淋巴细胞(Treg细胞)对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)的影响.方法 利用慢病毒载体介导,将鼠又状头螺旋转录因子(Foxp3)基因转导入Balb/c小鼠的CD4~+ CD25~-T淋巴细胞,即为基因工程Treg细胞.以Balb/c小鼠为供者,C57BL/6小鼠为受者,进行异基因骨髓移植,实验分4组进行:(1)工程Treg组经受鼠尾静脉输注供鼠骨髓细胞5×10~6个+脾细胞5×10~6个+基因工程Treg细胞5×10~6个;(2)移植对照组经受鼠尾静脉输注供鼠骨髓细胞5×10~6个+脾细胞5x10~6;(3)单纯照射组经受鼠尾静脉输注RPMI 1640培养液0.2ml;(4)空载体对照组经受鼠尾静脉输注供鼠骨髓细胞5×10~6个+脾细胞5×10~6个十空载体转导的CD4~+ CD25~-T 淋巴细胞5×10~6个.每天观察受鼠存活情况;记录GVHD的发生情况;各组均于小鼠濒死前取其肝脏、小肠、皮肤等组织,进行病理学观察;取长期存活(超过60d)的受鼠骨髓细胞,检测嵌合情况.结果 单纯照射组、移植对照组、工程Treg组和空载体对照组小鼠存活时间分别为(8.8±0.6)d、(36.7±2.5)d、(51.6±4.0)d和(34.1±2.3)d,工程Treg组小鼠存活时间明显长于其他各组,差异有统计学意义(P<0.05).移植对照组及空载体对照组小鼠肝脏、皮肤和小肠病理切片均存在GVHD病理改变,工程Treg组长期存活小鼠的肝脏、皮肤和小肠常规病理切片结构基本正常,未见GVHD病理表现,该组GVHD评分明显低于移植对照组及空载体对照组.结论 小鼠异基因骨髓移植时联合输注基因工程Treg细胞可有效减少GVHD的发生,减轻其严重程度.     

非清除性异基因骨髓移植后输注供者淋巴细胞诱导的GVHD和GVL效应

目的　探讨非清除性异基因骨髓移植后输注逐渐增量的异基因供者淋巴细胞 ,是否能在减轻移植物抗宿主病 (GVHD)的同时保留移植物抗白血病效应 (GVL)。方法　L6 15白血病小鼠(H 2 k)为受者 ,于接种白血病细胞后的第 3d接受60 Coγ射线全身照射 ,总剂量 5Gy ,照射当天移植BALB/c小鼠 (H 2 d)的骨髓细胞 (3× 10 7个 )和脾细胞 (1× 10 7个 ) ,移植后 2d腹腔注射环磷酰胺 2 0 0mg/kg ,并分别于移植后 14d或 14、2 1、2 8d输注 2× 10 7个和 1× 10 7、2× 10 7、4× 10 7个供鼠脾细胞。结果　未处理的对照组受鼠平均存活时间为 (14 .1± 1.1)d ,均死于白血病 ;移植后未接受供鼠脾细胞组的小鼠平均存活 (2 6 .2± 3.6 )d ,均死于白血病复发 ;移植后 14d接受供鼠脾细胞输注的小鼠平均存活 (2 9.3± 3.7)d ,均死于GVHD ;移植后输注逐渐增量的供鼠脾细胞者 ,平均存活 (5 9.4± 5 .6 )d ,6 0 %的受者存活超过 5 0d ,均未发生GVHD。结论　非清除性异基因骨髓移植后输注逐渐增量的异基因供者淋巴细胞可在减轻GVHD的同时保留抗白血病效应。     

心脏移植后骨髓腔内供者骨髓移植诱导供者特异性免疫低反应性再探讨

目的 再次探讨骨髓腔内供者骨髓移植诱导心脏移植后供者特异性免疫耐受的可行性.方法 2011年10月至2012年12月间,对3例心脏移植受者进行分期骨髓腔内供者骨髓移植.采用改良灌流法采集骨髓细胞,以增加骨髓细胞采集数目.应用抗胸腺球蛋白、氟达拉滨及巴利昔单抗进行预处理,以降低预处理不良反应.结果 应用改进灌流法采集的单核细胞数目由1.2×107/kg增加至5.8×107/kg(P＜0.01);CD34+细胞数目由2.38×105/k增加至6.7×105/kg(P＜0.01).骨髓移植后随访3个月,3例均无巨细胞病毒和真菌感染,未发生移植物抗宿主病等并发症.心内膜活检证实无排斥反应发生.体外混合淋巴细胞反应提示受者淋巴细胞对供者脾细胞呈免疫低反应性,而对无关第三者抗原仍维持良好的免疫活性.结论 改进的心脏移植后骨髓腔内供者骨髓移植诱导方案可降低骨髓移植后早期感染发生,提高了治疗的安全性,但仍可成功诱导供者特异性免疫低反应性.     

经抗CD158单克隆抗体修饰的供者自然杀伤细胞对受者树突状细胞的杀伤作用

目的 研究应用抗CD158a和CD158b单克隆抗体封闭供者杀伤细胞免疫球蛋白样受体(KIR)的抑制性受体KIR2DL1及KIR2DL3后,供者自然杀伤细胞(NK细胞)对受者树突状细胞(DC)的杀伤作用,及其减少移植物抗宿主病(GVHD)的作用.方法 对15例异基因全相合造血干细胞移植的供、受者进行HLA及KIR高分辨基因分析,采集供者骨髓及受者外周血单个核细胞,培养受者DC作为靶细胞,使用磁珠负选高纯度供者NK细胞作为效应细胞,用抗CD158a和CD158b单抗封闭供者KIR抑制性受体,并采用四甲基偶氮唑盐比色法分别检测抑制性KIR封闭前后NK细胞对靶细胞的杀伤作用.结果 在效靶比为10∶1下,供者NK细胞抑制性受体经抗体封闭后,对受者DC的杀伤活性明显提高,随着封闭抗体浓度的增加而增高,各浓度间比较,差异均有统计学意义(P＜0.05).当供、受者KIR配体匹配模式为宿主抗移植物(HVG)模式时,供者NK细胞对受者成熟DC的杀伤效果明显高于供、受者KIR基因全相合或移植物抗宿主(GVH)模式,在用不同浓度单抗封闭KIR抗体后,随着封闭抗体浓度的增加,杀伤效果增高更明显(P＜0.05).结论 利用抗CD158单抗封闭KIR可以提高供者来源NK细胞对受者DC的杀伤活性,输注异源反应性NK细胞可能作为一种新的治疗GVHD的方法.     

巴利昔单克隆抗体预防HLA不相合骨髓移植中移植物抗宿主病

目的研究巴利昔(商品名:舒莱)单克隆抗体体外对异基因反应T细胞和造血祖细胞的作用;探讨该抗体在预防HLA不相合骨髓移植中移植物抗宿主病(GVHD)的疗效。方法实验观察分两组。(1)巴利昔组:白血病患者72例,接受单倍型相合未去T细胞的骨髓移植,用巴利昔单克隆抗体预防GVHD;(2)对照组:2000年11月前进行上述相同骨髓移植的白血病患者15例,未用巴利昔单克隆抗体预防GVHD。用有限稀释方法检测巴利昔单抗对细胞毒介导的靶细胞前体(CTLp)的反应能力,在半固体造血祖细胞培养体系中检测巴利昔单抗对粒巨噬系祖细胞(CFUGM)、红系祖细胞(CFUE)及巨核系祖细胞(CFUMK)集落增殖的影响。结果巴利昔组72例骨髓移植后均取得造血重建,骨髓植活直接证据检测证实为完全供者造血,与对照组比较,差异无统计学意义。巴利昔组和对照组移植后急性Ⅱ～Ⅳ度GVHD发生率分别为12.5%(9例)和33.3%(5例),差异有统计学意义(P=0.045);两组的慢性广泛型GVHD发生率比较,差异无统计学意义。移植后18个月内动态观察免疫细胞重建,CD3、CD8、CD19、NK细胞及CD56细胞在12个月内可恢复至正常水平,CD4细胞在移植后18个月内恢复。随访中位数时间22个月,无病生存42例,KaplanMeier生存曲线分析,估计2年无病生存达58.3%。巴利昔组和对照组比较,无病存活和复发率差异无统计学意义。采用供者骨髓单个核细胞进行实验,显示巴利昔单抗能明显降低CTLp细胞生成,降低的数量在10～100倍,但不影响正常造血祖细胞CFUGM、BFUE和CFUMK集落的增殖。结论巴利昔单抗可选择性的限制和减少异基因反应T细胞,从而减少严重急性GVHD的发生率;白血病患者骨髓移植后造血重建未受影响,不增加复发率和感染率。     

The effect of G-CSF-stimulated donor marrow on engraftment and incidence of graft-versus-host disease in allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic bone marrow transplantation (allo-BMT). In an attempt to improve the results of HLA-identical sibling BMT, we investigated the effect of accelerating hemopoietic reconstitution and reducing acute GVHD (aGVHD) in allo-BMT receiving G-CSF-stimulated donor marrow and the preliminary biological mechanism. The donors of 30 patients (study group) with leukemia were given G-CSF 3-4 microg/kg/d for 7 doses prior to marrow harvest. The results of subsequent engraftment in the recipients were compared with those of 18 patients without G-CSF (control group). Five donors themselves were studied to assess the effects of G-CSF on the hematopoietic progenitor cells and lymphocyte subsets in the bone marrow (BM). We observed that the stimulated BM yielded higher numbers of nucleated cells as well as CFU-GM and CD34+ cells (p<0.01), and that hemopoietic reconstitution was accelerated. The median number of days of granulocyte count exceeding 0.5x10(9)/L and platelet count exceeding 20x10(9)/L was 16 (range 10-23 d) and 18.5 (range 13-31 d), respectively (control group: median 22 d, range 13-29 d and median 23 d, range 17-34 d; p=0.001). The incidence of grade II-IV severe aGVHD was very low, with only 1 case (3.3%) with acute grade II aGVHD limited to the skin in the study group. Five of 18 patients in the control group manifested grade II-IV severe aGVHD (27.8%, p=0.02). The number of T-lymphocyte subsets in the harvested BM using G-CSF stimulation was changed. In the G-CSF-stimulated marrow group, CD4+ decreased and CD8+ increased significantly (p=0.02). The changes of progenitor cells and T-lymphocyte subsets in donors' BM from pre- and post-G-CSF stimulation showed that the percentage of CD4+ reduced (p=0.04) and that of CD8+ increased (p=0.06), while that of CD34+ also increased (p=0.002). The incidence of chronic GVHD and relapse had no significant difference between both groups. These results indicate that allo-BMT in BM G-CSF priming can accelerate engraftment and minimize the incidence of severe aGVHD. There is a trend in favor of improved transplantation-related mortality.     

Acceleration of the hemopoietic reconstitution in mice undergoing bone marrow transplantation by recombinant human granulocyte colony-stimulating factor.

We have investigated the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 micrograms/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT.     

Enhanced antitumor effects of bone marrow transplantation in combination with fibroblast-mediated IL-2 and IL-3 gene therapy.

BACKGROUND: Bone marrow transplantation (BMT) and gene therapy are potent approaches to the recovery of bone marrow depression and induction of antitumor immunity after chemotherapy for the treatment of malignancies. In the present study, enhanced antitumor effect of BMT in combination with fibroblast-mediated interleukin (IL)-2 and IL-3 gene therapy was observed in tumor-bearing mice after chemotherapy. METHODS: BALB/c mice were inoculated s.c. with J558L plasmacytoma cells and injected i.p. with cyclophosphamide 300 mg/kg 3 days later. 24 hours after chemotherapy syngeneic bone marrow cells in combination with NIH3T3 fibroblast cells engineered to produce IL-2 (NIH3T3-IL-2) and/or NIH3T3 cells engineered to produce IL-3 (NIH3T3-IL-3) were implanted into the tumor-bearing mice. RESULTS: BMT in combination with implantation of either NIH3T3-IL-2 or NIH3T3-IL-3 cells exerted significant inhibition on the growth of J558L tumors and prolonged the survival period of the tumor-bearing mice as compared with the treatments with Hanks solution, BMT alone, or BMT plus implantation of NIH3T3 cells transduced with Neo gene. Synergistic antitumor effect was observed in mice after combined BMT and cytokine gene therapy. The cytotoxicities of natural killer cells, cytotoxic T lymphocytes, and macrophages in mice increased markedly after the combined treatment. Recovery of CFU-GM, CFU-MK and CFU-E formation in mice after combined therapy was accelerated obviously in mice after combined therapy. CONCLUSIONS: BMT in combination with fibroblast-mediated IL-2 and IL-3 gene therapy elicited augmented antitumor effects synergistically in tumor-bearing mice after chemotherapy mainly through induction of antitumor immune response and accelerated recovery of hematopoiesis.     

粒细胞集落刺激因子和干细胞因子对骨髓单个核细胞的动员效率及其机制

目的 联合应用粒细胞集落刺激因子(G-CSF)和干细胞因子(SCF)动员骨髓单个核细胞,评价其动员效果,探讨CXCL12/CXCR4信号通路在骨髓单个核细胞动员中的作用及机制.方法 将昆明小鼠随机分为两组,治疗组皮下注射重组鼠G-CSF 100μg/(kg·d)和重组鼠SCF25μg/(kg·d),连续使用5d;对照组皮下注射等剂量的生理盐水.每组于不同时间点取小鼠骨髓,分离培养骨髓单个核细胞,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪分选CD34+ CXCR4+单个核细胞(MNCs);应用酶联免疫吸附试验(ELISA)法测定骨髓细胞外液CXCL12a的水平;采用逆转录-聚合酶链反应(RT-PCR)检测骨髓CXCL12 mRNA表达变化.结果 应用G-CSF/SCF后,骨髓及外周血中单个核细胞计数较对照组明显增加(P<0.01),CFU-F形成能力显著增强(P<0.05);流式分选表明CD34+ CXCR4+细胞占骨髓CD34+单个核细胞总数的(44.6±8.7)%;RT-PCR和EUSA检测示骨髓CXCL12 mRNA表达下降,骨髓细胞外液CXCL12蛋白也显著下降,两者变化一致.结论 G-CSF/SCF可有效地诱导骨髓单个核细胞动员,其机制可能是通过破坏骨髓CXCL12/CXCR4信号通路平衡,下调CXCL12/CXCR4间相互作用,以促进骨髓单个核细胞动员.     

Effects of flt-3 ligand in combination with TPO on the expansion of megakaryocytic progenitors

As an early acting growth factor, flt-3 ligand (FL) promotes the ex vivo expansion of hematopoietic stem and progenitor cells. The effect and mechanism of FL on the development of the megakaryocytic lineage remain unclear. In this study, we compared the effects of FL and stem cell factor (SCF) in combination with other megakaryocyte-promoting cytokines on the differentiation and proliferation of megakaryocytic progenitors and investigated the expression of flt-3 receptors on megakaryocytic cell lines. In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. Using murine bone marrow (BM) cells, we demonstrated that FL at a range of 0-100 ng/ml had no significant mitogenic effect on CFU-MK formation. TPO increased CFU-MK (p < 0.001) but the effect was not significantly modified by FL. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). FL alone did not stimulate megakaryocytopoiesis, possibly due to the lack of receptor expression on megakaryocytes. The effect of FL in augmenting the expansion of CFU-MK in liquid culture might be due to the early action of FL at the pluripotent stem cell stage.