HLA位点不全相合的异基因干细胞移植治疗白血病的临床分析

目的：探讨HLA不完全相合的造血干细胞移植治疗白血病的新方法.方法：将8例白血病患者接受FBC预处理方案后行异基因粒细胞集落刺激因子（G-CSF）动员的骨髓和外周血干细胞联合移植，观察造血重建和移植相关的并发症情况.结果：1例2位点不合患者植入失败，其余7例完全植入，白细胞恢复时间13.5 d，血小板恢复15.1 d，7例患者Ⅰ～Ⅱ度急性移植物抗宿主病（GVHD）发生率57.1%（4/7），局限性慢性GVHD发生率83.3%，未出现严重的心、肝和肺脏并发症.移植后6个月生存率62.5%.结论：用FBC预处理方案和GCSF动员后的骨髓和外周血干细胞联合移植的方法对于HLA不相合的移植安全有效。     

HLA单倍相合造血干细胞移植治疗恶性血液病的临床研究

目的 探讨人类白细胞抗原(HLA)单倍相合造血干细胞移植(HSCT)治疗恶性血液病的疗效和主要并发症.方法 对2004年7月至2006年12月第三军医大学附属新桥医院收治的35例恶性血液病患者进行HLA单倍相合亲缘供者HSCT.采用延长、强化联合免疫抑制促进植入、抗胸腺细胞球蛋白(ATG)加强预防移植物抗宿主病(GVHD)、粒细胞集落刺激因子(G-CSF)动员的骨髓(BM)加外周血干细胞(PBSC)混合移植方案.结果 所有患者均重建供者造血.18例(51.4%)发生急性GVHD(aGVHD),其中Ⅰ度8例,Ⅱ度5例,Ⅲ度3例,Ⅳ度2例,Ⅱ～Ⅳ度aGVHD累积发生率为28.6%.12例(34.3%)发生慢性CVHD(cGVHD),均为局限性.23例患者存活,总存活率为65.7%,2年无病存活率(DFS)为62.9%.12例患者死亡,7例死于复发,5例死于移植相关合并症,其中肺部感染2例,Ⅳ度GVHD 2例,巨细胞病毒(CMV)感染1例.结论 随着预处理方案、GVHD预防方案及移植物成分的优化,HLA单倍相合造血干细胞移植的疗效明显改善,已成为治疗恶性血液病的重要方法.     

低剂量ATG用于父母HLA 10/10相合造血干细胞移植患者的临床分析

目的:探讨父母HLA 10/10相合不去除T细胞的外周血造血干细胞移植治疗恶性血液病的植入情况、疗效和相关并发症。方法:预处理采用改良的Bu/Cy方案,用低剂量的兔抗人T淋巴细胞球蛋白、环孢素A、吗替麦考酚酯和短程甲氨蝶呤预防移植物抗宿主病(GVHD)。回输G-CSF动员的外周血干细胞,输注单个核细胞中位数为7.830(7.320~10.004)×108/kg,CD34+细胞中位数为4.59(3.68~5.32)×106/kg。结果:4例患者均达完全供者植入,粒系植入时间为16(11~16)d,血小板植入时间为22(11~29)d。发生Ⅱ度急性GVHD1例,发生率25%。发生慢性GVHD 3例,广泛型1例。中位随访267(178~1903)d,死亡1例(非复发死亡),复发1例(为高危患者),无复发存活2例。结论:低剂量ATG用于父母HLA 10/10相合供者异基因造血干细胞移植治疗高危恶性血液病是比较安全、有效的方案。     

异基因造血干细胞移植治疗白血病20例临床观察

目的探讨不同类型异基因造血干细胞移植(allo-HSCT)治疗白血病的疗效、造血重建及存活情况。方法哈尔滨血液病肿瘤研究所2003年3月至2006年7月进行异基因造血干细胞移植的白血病患者20例,其中同胞间人类白细胞抗原(HLA)相合的异基因外周血造血干细胞移植(allo-PBSCT)12例,无亲缘关系HLA不全相合脐血移植(UCBT)4例,无关供者的异基因外周血干细胞移植(其中1例HLA-CW位点亚型不合)3例,无关供者骨髓移植1例(经过去除红细胞处理)。结果19/20受者获造血重建,UCBT患者造血重建速度较HLA相合的同胞allo-PBSCT慢,异基因造血干细胞移植后20例患者中发生急性移植物抗宿主病(aGVHD)7例,其中4例Ⅰ~Ⅱ度,3例Ⅲ~Ⅳ度。3例患者死于复发,3例死于移植物抗宿主病(GVHD),另15例至今仍无病存活,中位存活时间30(1~41)个月。结论allo-HSCT是目前治愈白血病的有效方法,对于无同胞HLA相合的供者,选择较高细胞数量,HLA1~2个位点不合的UCBT仍然有效、可行。     

非清髓造血干细胞移植治疗再生障碍性贫血

目的:探讨非清髓性造血干细胞移植(NST)治疗再生障碍性贫血(再障)的方法及疗效。方法:采用非清髓预处理方案进行造血干细胞移植治疗再生障碍性贫血2例。1例为同胞间HLA配型6个位点完全相合的异基因外周血造血干细胞移植,另1例为同胞间HLA配型6个位点完全相合的脐血移植。预处理方案主要由抗胸腺细胞球蛋白(ATG)和环磷酰胺组成。用环孢素A和霉酚酸酯(MMF)预防移植物抗宿主病(GVHD)。结果:2例患者均获造血重建(分别为 5及 9d),2例均未发生GVHD。1例患者在治疗期间未出现感染表现,另1例患者出现CMV感染,给予更昔洛韦病情得以完全控制。2例分别无病生存8及17个月。结论:非清髓造血干细胞移植简便安全,并发症少,疗效好,为治疗再生障碍性贫血有效方法。     

HLA配型相合的造血干细胞移植治疗重型再生障碍性贫血的临床研究

目的 评价HLA配型相合的异基因造血干细胞移植(allo-HSCT)治疗重型再生障碍性贫血(SAA)的疗效.方法 2000年1月至2008年11月采用allo-HSCT治疗SAA患者20例,其中同胞相合移植17例,非血缘关系移植3例.预处理采用环磷酰胺(Cy)50 mg·kg~(-1)·d~(-1)4 d加抗淋巴细胞免疫球蛋白(ATG)2.5 mg·kg~(-1)·d~(-1)或20 mg·kg~(-1)·d~(-1) d.移植物抗宿主病(GVHD)的预防方案为经典的环孢素A(CsA)联合短程甲氨蝶呤(MTX)及霉酚酸酯(MMF).同胞供者采集经重组人粒细胞集落刺激因子(G-CSF)动员的骨髓及外周血干细胞,非血缘供者单纯采集外周血干细胞.结果 回输单个核细胞中位数为7.89(4.00-14.21)×10~(8)/kg,所有患者均获供者造血重建,粒细胞植活中位时间14(11～20)d;血小板植活中位时间12(8～108)d.但1例患者发生晚期排斥,行另一供者二次移植后植活.21例次移植后共发生6例次急性GVHD(I度3例,Ⅱ度皮肤3例),发生率16%.19例生存期>100 d的患者中有7例发生慢性GVHD,其中4例为局限型,3例为广泛型.截至2009年2月28 日,经过中位18(2.0～106.8)个月的随访,共有17例患者无病生存,总生存率为82.5%.结论 采用Cy+ATG的预处理方案对SAA患者进行HLA配型相合HSCT,植活率高,可以获得良好的疗效.     

血液肿瘤病人HLA匹配的骨髓移植与外周血干细胞移植的疗效对比分析

使用粒细胞集落刺激因子(G-CSF)动员获得的异基因外周血造血干细胞进行移植(PBCT),其造血功能的重建作用明显快于骨髓移植(BMT),但两者急、慢性移植物抗宿主病(GVHD)的发生率、复发率、无病存活率是否存在差异还有待研究。作者对PBCT和BMT进行了比较。     

含全身照射预处理方案行HLA不相合非去T造血干细胞移植治疗白血病8例临床分析

目的探讨采用全身照射(TBI)预处理方案行人类白细胞抗原(HLA)配型不相合亲缘供者非去T异基因造血干细胞移植(allo-HSCT)治疗白血病的疗效。方法2002年4月至2007年1月北京大学血液病研究所8例采用TBI预处理方案行HLA不合非去T亲缘供者allo-HSCT的白血病患者,其中急性髓性白血病(AML)3例,急性淋巴细胞性白血病(ALL)4例,慢性粒细胞白血病1例;预处理方案采用TBI加环磷酰胺(CY)方案4例,TBI加氟达拉滨(FLU)方案4例;干细胞来源包括骨髓和外周血造血干细胞移植6例,外周血造血干细胞移植(PBSCT)2例;移植物抗宿主病(GVHD)预防采用经典的环孢素A(CsA) 霉酚酸酯(MMF) 短程甲氨蝶呤(MTX)方案。结果8例供者采集单个核细胞(MNC)中位数为7.39(6.27~12.46)×108/kg,粒细胞植入中位时间11(11~13)d,血小板植入中位时间13(11~21)d。5例发生Ⅰ~Ⅱ度急性GVHD,2例出现慢性广泛性GVHD,无严重急性GVHD或因GVHD死亡病例。中位随访时间9(3~53)个月,除1例复发存活外,其余病例无病存活。结论对于HLA不相合异基因造血干细胞移植,TBI方案是一种比较安全、有效的非去T预处理方案,对于高危和二次移植患者同样有效。     

异基因造血干细胞移植治疗高龄恶性血液病的疗效分析

目的探讨异基因造血干细胞移植治疗年龄较大的白血病患者安全性和疗效。方法采用HSCT治疗10例45—63岁恶性血液病患者,HLA6／6位点相合8例,5／6位点相合2例。以清髓性方案预处理4例,减低强度方案预处理6例。HLA不全相合患者加用ATG。采用环孢素联合霉酚酸酯预防移植物抗宿主病（GVHD）。结果本组10例受者均获造血重建。中性粒细胞绝对计数≥0．5×10^9／L,血小板≥20×10^9／L的中位时间分别为移植后12（9～16）天和15（12～21）天。3例发生了急性GVHD（30％）,Ⅱ度以上2例。可评估的9例患者中2例出现了慢性GVHD（22％）。复发2例,死亡3例。可评估的2年无病生存率为70％。结论造血干细胞移植对于高龄白血病患者是一种有效、安全的根治疗法。     

非清髓无关供体外周血干细胞移植治疗重型再生障碍性贫血

目的 探讨无关供体造血干细胞移植治疗重型再生障碍性贫血(SAA)的方法 和疗效.方法 对1例SAA的患者进行了无关供体HLA高分辨4/6相合的外周血干细胞移植.采用环磷酰胺(100 mg/kg) 氟达拉宾(150 mg/m2) 抗人淋巴细胞球蛋白(100 mg/kg)的非清髓性预处理后,回输粒细胞集落刺激因子(G-CSF)动员的外周血干细胞,共输注单个核细胞(MNC)6.77×108/kg,CD 34细胞1.95×106/kg.预防移植物抗宿主病(GVHD)采用环胞菌素A(CsA)联合短疗程甲氨蝶呤(MTX)的基础上加用霉酚酸酯(MMF)的方案.结果 患者移植后造血恢复顺利,于移植后第6天WBC植入,第8天PLT植入,第30天行患者骨髓STR-PCR检测显示为完全供者的基因型,第150天血型转变为供者型(O→A).未发生急性GVHD(aGVHD)及慢性GVHD(cGVHD),随访至移植后8个月,造血功能恢复良好,仍在继续随访中.结论 以氟达拉宾、环磷酰胺和抗人淋巴细胞球蛋白组成的非清髓性预处理方案用于无关供体外周血干细胞移植治疗SAA,能够获得稳定的植入,且并发症少,是有效移植方法之一.     

Glucose-responsive insulin-producing cells from stem cells

Recent success with immunosuppression following islet cell transplantation offers hope that a cell transplantation treatment for type 1 (juvenile) diabetes may be possible if sufficient quantities of safe and effective cells can be produced. For the treatment of type 1 diabetes, the two therapeutically essential functions are the ability to monitor blood glucose levels and the production of corresponding and sufficient levels of mature insulin to maintain glycemic control. Stem cells can replicate themselves and produce cells that take on more specialized functions. If a source of stem cells capable of yielding glucose-responsive insulin-producing (GRIP) cells can be identified, then transplantation-based treatment for type 1 diabetes may become widely available. Currently, stem cells from embryonic and adult sources are being investigated for their ability to proliferate and differentiate into cells with GRIP function. Human embryonic pluripotent stem cells, commonly referred to as embryonic stem (ES) cells and embryonic germ (EG) cells, have received significant attention owing to their broad capacity to differentiate and ability to proliferate well in culture. Their application to diabetes research is of particular promise, as it has been demonstrated that mouse ES cells are capable of producing cells able to normalize glucose levels of diabetic mice, and human ES cells can differentiate into cells capable of insulin production. Cells with GRIP function have also been derived from stem cells residing in adult organisms, here referred to as endogenous stem cell sources. Independent of source, stem cells capable of producing cells with GRIP function may provide a widely available cell transplantation treatment for type 1 diabetes.     

Insulin expressing cells from differentiated embryonic stem cells are not beta cells

Aim/hypothesis Embryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes.Methods ES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks.Results Insulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state.Conclusions/Interpretation Our studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.Abbreviations ES Embryonic stem - LIF leukemia inhibitory factor - ITSF insulin-transferrin-selenite-fibronectin.Bleackley and Korbutt laboratories contributed equally to this paper     

DC与CIK或LAK联合对结肠癌细胞株SW480杀伤作用的研究

[目的]研究树突状细胞(DC)联合细胞因子诱导或未诱导的杀伤细胞(CIK)或淋巴因子激活的杀伤细胞(LAK)对结肠癌细胞株SW480的杀伤活性.提供DC联合CIK或LAK治疗结肠癌的实验依据.[方法]取人外周血分离出单个核细胞(PBMNC),诱导生成DC、CIK、LAK细胞;流式细胞仪检测DC经SW480肿瘤抗原冲击后的表型变化;以CIK+DC细胞、CIK细胞、LAK+DC细胞及LAK细胞作为效应细胞,SW480为靶细胞,以15∶1、30∶1、45∶1为效靶比,LDH释放法测定细胞杀伤试验活性;ELISA检测杀伤试验中干扰素γ(IFN-γ)、白细胞介素2(IL-2)、IL-12、IL-17的分泌水平.[结果]流式细胞仪检测DC经SW480肿瘤抗原冲击后,其表面分子HLA-DR、CD40、CD80和CD86表达分别平均为90.23％、73.68％、85.96％、57.55％,与未经肿瘤抗原冲击DC比较,DC成熟的表面标志分子表达明显增加(P＜0.01).相同效靶比下,CIK+DC细胞组对SW480的杀伤作用最强,明显高于其他细胞组(P＜0.01);CIK+ DC细胞组在效靶比为45∶1时,杀伤活性最强(P＜0.01);单独CIK细胞组的杀伤活性明显高于LAK+DC细胞组(P＜0.01);LAK+ DC细胞组的杀伤活性明显高于单独LAK细胞组(P＜0.01).效靶比为45∶1时,各杀伤试验细胞组上清液中IFN-γ、IL-2、IL-12、IL-17的分泌量,CIK+DC细胞组的IFN-γ、IL-12的分泌量显著高于其他细胞组(P＜0.05);LAK+DC、单独LAK细胞组IL-2的分泌量明显高于CIK+DC、单独CIK细胞组(P＜0.05);单独CIK细胞组IFN-γ的分泌量明显高于LAK+DC、单独LAK细胞组(P＜0.05).[结论]CIK+DC细胞组对SW480的杀伤活性明显强于单独CIK、LAK+ DC组、单独LAK细胞组.其机制可能是,SW480抗原致敏的DC分泌IFN-γ、IL-12等刺激、诱导CIK细胞的活化和增殖,明显增强CIK细胞杀伤SW480的活性.     

树突状细胞联合细胞因子诱导的杀伤细胞对胃癌细胞杀伤作用

目的探讨树突状细胞联合细胞因子诱导的杀伤细胞对胃癌细胞的杀伤作用。方法采用胃癌患者自身血液中单个核细胞(peripheral blood mononuclear cells,PBMC),经体外诱导分别扩增出DC和CIK细胞,二者共同培养后,利用MTT法检测DC细胞联合CIK细胞体外杀伤人胃癌细胞株(MNK-45、MNK-28、SG-7901)的活性。结果DC与CIK细胞共培养后得到的细胞群高表达CD3 CD56 ,平均值达到(56.74±7.63)%。通过彼此相互作用诱导出的细胞群体对胃癌细胞株MNK-45、MNK-28、SG-7901有杀伤作用,且杀伤活性随着效靶比的增加而增强。结论DC与CIK细胞共培养后有很强的增殖能力,对胃癌细胞具有杀伤活性,且其杀伤作用与胃癌细胞类型无相关性。     

Endothelial cells as cytotoxic effector cells: cytokine-activated rat islet endothelial cells lyse syngeneic islet cells via nitric oxide

Summary In vivo, each beta cell is located in proximity to at least one capillary islet endothelial cell. Rat aorta and islet endothelial cells can be activated in vitro to express inducible nitric oxide synthase by a cytokine mixture of tumour necrosis factor-α, gamma-interferon, and interleukin-1β and to produce high concentrations of nitric oxide. We have performed co-culture experiments with rat islet endothelial cells together with isolated syngeneic islet cells at low target : effector ratios with or without previous cytokine challenge of endothelial cultures. Co-cultures were always free of exogenous cytokines, which were removed prior to addition of islet cells. We found that pre-activated, in contrast to resident islet endothelial cells, at a target : effector ratio as low as 1 : 1 almost completely lysed syngeneic beta and non-beta cells within 24 h of co-culture. Lysis by pre-activated islet endothelial cells was found to be preceded by DNA damage found in 46 % of islet cells after 8 h of co-culture with pre-activated vs 7 % with resting islet endothelial cells. Lysis was blocked to control levels in the presence of the nitric oxide synthase inhibitor N^G-methyl-L-arginine. With the results presented here, we demonstrate for the first time, that activated endothelial lining cells can express effector cell activity and thus can contribute to local tissue destruction, especially in organs that are densely capillarized such as pancreatic islets. [Diabetologia (1997) 40: 150–155] Received: 2 September 1996 and in revised form: 24 October 1996     

Dendritic cells: specialized antigen presenting cells

Renewing interest in cancer immunotherapy reflects the excellent results that have been obtained in animal models and the promising results in early clinical trails with dendritic cell (DC) based approaches. The central role that DCs play in the initiation of an immune response raises the possibility of using them to trigger specific anti-tumor immunity. In addition, deeper knowledge of DC biology will allow better understanding of the mechanism(s) underlying allergic and autoimmune diseases as well as tolerance phenomena. These crucial issues were critically reviewed during a workshop organized by the Italian Society for Experimental Hematology in Florence, Italy, on March 18th, 1999. The chairmen have prepared this report for the readers of Haematologica.