自体CD34~+细胞移植治疗晚期肿瘤

目的　采用CD34+ 细胞体外分选技术对晚期肿瘤患者进行自体CD34+ 细胞移植 ,以降低自体移植后肿瘤复发率。方法　对 15例Ⅲ～Ⅵ期肿瘤患者 (多发性骨髓瘤 11例 ,乳癌 2例 ,非霍奇金淋巴瘤和髓母细胞瘤各 1例 )采用CliniMACS临床型细胞富集仪 ,利用磁性分选技术收集CD34+和CD34-细胞组分 ,患者于预处理后 ,输注分选后的CD34+ 细胞。结果　CD34+ 细胞体外纯化富集可使CD34-细胞获得 2 .0～ 5 .0个对数的去除 ;回输CD34+ 细胞中位数为 2 .4× 10 6/kg,CD34+ 细胞回收率为 6 4 % ,纯度为 98.2 % ;移植后白细胞恢复至 >1.0× 10 9/L和血小板 >2 0× 10 9/L的天数 (中位数 )分别为 14d和 13d。患者总体生存率 6 6 .7% (10 / 15 ) ,无疾病生存率 5 3.3% (8/ 15 )。结论　CD34+ 细胞移植后获得迅速、稳定的造血重建。体外CD34+ 细胞纯化富集后移植可望提高晚期肿瘤患者自体移植疗效。     

自体外周血CD34^＋造血干细胞移植治疗多发性骨髓瘤

[目的]探讨自体外周血CD+34造血干细胞移植治疗多发性骨髓瘤的可行性。[方法]应用CliniMACS系统分选纯化 患者外周血CD+34造血干细胞,用流式细胞仪检测分选纯化 结果。选马法兰200mg/m2作为预处理方案。[结果]分选纯化结 果,单个核细胞数为3.7136×108,活性率96.6％,CD+34细胞数 3.68018×108,即5.52×106/kg。移植后患者骨髓像获得完全缓 解。移植相关并发症少且易于控制。[结论]自体外周血CD+34 造血干细胞移植治疗多发性骨髓瘤是安全、有效、可行的 。     

CD4^＋T和CD8^＋T细胞的新功能

Ｔ细胞为抗癌免疫反应的核心细胞，成熟Ｔ细胞为αβＴＣＲ＾＋Ｔ细胞，主要为ＣＤ４＾＋和ＣＤ８＾＋两大亚群。ＣＤ４＾＋Ｔ细胞不仅在初始免疫反应中行使强有力的调节作用，在抗癌免疫记忆反应中更为重要且可参与靶细胞的直接杀伤。ＣＤ８＾＋Ｔ细胞除行使杀伤效应外，亦具有强烈的调节效应，并可类似于Ｔｈ１／Ｔｈ２而分化出Ｔｃ１／Ｔｃ２，从而参与针对癌症，病毒，胞内菌和自身免疫病的调节。     

Effect of Homoharringtonine on Bone Morrow CD34^＋CD7^＋ Cells in Chronic Granulocytic Leukemia

Objective： To explore the effect of homoharringtonine （HHT） on bone morrow CD34^＋CD7^＋ cells in chronic granulocytic leukemia （CGL）. Methods： The changes of bone morrow CD34^＋CD7^＋ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34^＋CD7^＋ cells treated with HHT in vitro were studied. Results： The proportion of CD34^＋CD7^＋ cells in CGL （0.145±0.021） was higher than that of normal control （0.052±0.013）. The proportion of CD34^＋CD7^＋ cells in patients who got cytogenetic responses to HHT （0.072±0.020） decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, （0.137±0.023）. the proliferation of CD34^＋ cells was inhibited and the proportion of CD34^＋CD7^＋ cells decreased after cultured with HHT （0.134 in 24 h, 0.126 in 48 h and 0.102 in 72）. The apoptosis rate of CD34^＋CD7^＋ cells was higher than that in CD34^＋CDT cells （35.39%±4.39% versus 24.57%±4.01%, P〈0.05） 72 h after culture with HHT. Conclusion： The proportion of CD34^＋CD7^＋ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34^＋CD7^＋ cells.     

纯化的自体CD34+细胞移植治疗恶性淋巴瘤

自体造血干细胞移植是NHL患者取得长期生存的重要治疗方法之一。但是,自体移植物中肿瘤细胞的污染,成为auto-HSCT后NHL复发的主要原因之一：因此,体外净化去除移植物中可能存在的残留肿瘤细胞,对于降低auto-HSCT后的复发率具有重要意义。本文就NHL自体造血干细胞移植中,CD34^ 细胞分选的机制、方法以及CD34阳性细胞移植在淋巴瘤治疗中的疗效做一简单综述。     

肿瘤患者CD4^＋CD25^＋调节性T细胞流式细胞术分析

目的：分析比较肿瘤患者和健康人外周血CD4^＋CD25^＋调节性T细胞的特点,为肿瘤免疫学研究及治疗探索新方法。方法：收集并分离30例肿瘤患者和32例健康人的外周血单个核细胞（PBMCs）,用荧光标记的抗人CD4及抗人CD25单抗标记肿瘤患者和健康人PBMCs细胞,FCM检测CD4^＋CD25^＋Treg细胞,分析CD4^＋CD25^＋Treg细胞在肿瘤患者和健康人中的差别。结果：肿瘤患者的CD4^＋CD25^＋Treg细胞百分数明显高于健康人（年龄〈55者62．4vs40．4;年龄≥55者53．1vs31．0,P〈0．05）。结论：肿瘤患者的CD4^＋CD25^＋Treg细胞高于健康对照,为肿瘤免疫治疗提供新策略,通过删除CD4^＋CD25^＋Treg细胞,有可能增强抗瘤免疫反应。     

脐血CD34^＋细胞体外扩增及树突状细胞诱导的实验研究

目的 研究脐血CD34^＋细胞在体外经造血细胞生长因子扩增后,诱导树突状细胞（DC）并观察该类DC在抗肿瘤免疫中的作用。方法 Ficoll分离脐血单个核细胞,免疫微磁珠法分离纯化CD34^ 细胞,以干细胞因子、flt3配体、白细胞介素－3和红细胞生成素体外扩增2周,诱导DC生成,观察肿瘤抗原负载后诱导特异性细胞毒T淋巴细胞生成和对肿瘤细胞杀伤作用。结果（1）脐血经免疫微磁珠法分离可获得高纯高的CD34^ 细胞;（2）2周体外扩增后,细胞数显著增加达150倍;体外集落试验证实该类细胞可形成粒单细胞集落形成单位／（CFU－GM）;（3）扩增的细胞可成功诱导成DC,并具有活化异体淋巴细胞的功能,负载肿瘤抗原后能诱导发生肿瘤特异性淋巴细胞,并可特异性杀伤Daudi肿瘤细胞。结论 脐血来源CD34^ 细胞在体外能成功地扩增,并诱导产生功能性DC。     

CD34^＋细胞的体外定向诱导分化

目的：研究CD34^ 细胞的体外定向诱导分化。方法：利用吸附单克隆抗体-磁珠分离系统（MACS）从外周血中分离CD34^ 细胞,加入白细胞介素-3（IL-3）、粒细胞-单核巨噬细胞集落刺激因子（GM-CSF）及G-CSF培养3天及7天后取出用于细胞总数,CD34^ 细胞比例及粒系祖细胞检测。结果：CD34^ 细胞在IL-3、G-CSF、GM-CSF刺激下3天细胞总数扩增51倍,7天细胞总数扩增110倍,IL-3、G-CSF、CM-CSF可使粒=单核巨噬系祖细胞（CFU-GM）在3天和7天时分别比对照组增加8.44wuk t 35.08倍。结论：含有IL-3、G-CSF、GM-CSF的细胞因子组合。可诱导CD34^ 细胞定向生成大量的粒系祖细胞和成熟粒细胞,输注后将可使患者白细胞得以迅速恢复,减少感染的发生率。     

用纯化CD+34细胞进行HLA半相合异基因造血干细胞移植治疗慢性粒细胞白血病1例

异基因造血干细胞移植是公认的根治血液系统恶性肿瘤的治疗方法,但由于HLA完全相合的合适供者的来源较为困难,成为限制其临床上广泛开展的重要原因,我们运用CD+34阳性选择的方法进行1例HLA半相合的异基因造血干细胞移植,现报告如下.     

应用C1iniMACS细胞分选系统纯化自体外周血干细胞的探讨

目的 探讨应用CliniMACS细胞分选系统纯化自体外周血干细胞的效果。方法 4例非霍奇金淋巴瘤患者,经Vp-16+G-CSF动员后,采集外周血中造血干细胞,并用CliniMACS细胞分选系统作干细胞纯化分选,3例患者作2次采集后行1次分选,1例患者经3次采集后作2次分选。通过单个核细胞计数及流式细胞术测定CD34+细胞,分析纯化效果。结果 4例患者进行7例次干细胞采集,循环量6000～8 000ml,采集前体内CD34+细胞数为每千克体重(12.77±11.21)×106,经采集得到CD34+细胞数为每千克体重(4.70±4.17)×106,CD34+细胞采集效率为(40.91±33.35)％,对5例次磁珠分选结果,标记前CD34+细胞数为每千克体重(6.07±3.18)×106,分选后获得的CD34+细胞数为每千克体重(4.03±2.28)×106,CD34+细胞回收率为(64.19±13.35)％。终产物细胞悬液量为40～45 ml,CD34+细胞纯度为(83.18±6.76)％。结论 应用CliniMACS细胞分选系统获得纯化的自体造血干细胞,可应用于支持大剂量化疗治疗实体肿瘤。     

CD34+ CD90+ cells and late hematopoietic reconstitution after autologous peripheral blood stem cell transplantation

Autologous peripheral blood stem cell transplantation (PBSCT) has been demonstrated to result in rapid, stable long-term engraftment. However, there has been considerable debate concerning the cells responsible for early and late hematopoietic reconstitution after PBSCT. Recently, CD34⁺ hematopoietic stem and progenitor cells have been clearly divided into two subpopulations by flow cytometry; namely undifferentiated pluripotent stem cells and differentiated committed progenitor cells. However, only a few studies have defined which subset contained in graft products might be the most predictive for late hematopoietic reconstitution after PBSCT. In this review, we present updated information regarding the relationships between the number of infused CD34⁺ cells or their immature subsets such as CD34⁺CD90⁺ cells and the late hematopoietic reconstitution after PBSCT, and discuss the threshold dose of CD34⁺CD90⁺ cells required for sustained long-term engraftment.     

Long-term follow-up of gene-marked CD34+ cells after autologous stem cell transplantation for multiple myeloma

Gene marking can be used to investigate if progenitor cells harvested from patients are contaminated with tumorigenic cells. It can also provide information about the contribution of hematopoietic stem cells to long-term engraftment and about long-term transgene expression from integrated retroviral vectors. In order to study autologous-infused cell contribution to relapse as well as the long-term persistence of the transgene in hematopoietic cells following autologous bone marrow (BM) transplantation for multiple myeloma, we genetically marked autologous CD34+ enriched BM or peripheral blood cell grafts of eight myeloma patients using retroviral vectors. Six patients were subsequently transplanted with the marked graft and followed with regular time points of analysis. Briefly, mononuclear cells were harvested by leukapheresis during 2-4 consecutive days following priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF. The CD34+ cells separated on Cellpro ceprate avidin-biotin columns were exposed to the G1Na vector coding for neomycin resistance gene at a ratio of five vector particles per cell at three consecutive time points achieving an average transduction efficacy of 2% (0.43-5.1%). The patients were transplanted with a mixture of transduced cells and un-manipulated graft. Vector integration and transgene expression were analyzed by colony assays and polymerase chain reaction. The transgene could be detected for up to 5 years post-transplant in normal BM cells, even in remission following relapse and no side effects related to retroviral gene transfer were observed. There were no marked myeloma cells observed in the patients either in remission or in relapsing disease, which indicates that contribution of infused cells to relapse is unlikely.     

自体外周血造血干细胞移植过程中淋巴细胞亚群和CD34+细胞变化的动态研究

目的动态观察动员、采集过程中外周血及造血干细胞中的淋巴细胞亚群和造血干细胞含量变化,及时指导临床选择最佳采集时机。方法 采用流式细胞术(FCM)测定大剂量化疗(HDC)联合重组人粒细胞集落刺激因子(rhG-CSF)对47例恶性实体瘤患者自体外周血造血干细胞移植(APBSCT)过程中,外周血和外周血造血干细胞(PBPC)中的淋巴细胞亚群CD3、CD4、CD8、NK、CD19和造血干细胞CD34+及其亚群CD34+CD38-、CD34+ Thy1+、AC133+细胞含量的变化,同时用体外集落培养的方法评价干细胞克隆形成能力。结果 动员后外周血淋巴细胞亚群CD3、CD4、CD8、NK和CD19细胞含量均低于动员前,其中CD3、CD8含量明显低于基础状态(P=0.007,P=0.016),而动员后外周血中的CD34及其亚群含量均明显高于动员前(P<0.05)。动员后中位时间第16天(15～17 d)外周血中的CD34含量达到最高峰,且第1次采集物中的造血干细胞含量最高。PBPC中除CD4、CD4/CD8明显低于外周血外(P<0.000 5),其他淋巴细胞亚群含量与外周血比较无明显改变。外周血单个核细胞中CD34+细胞含量与PBPC中CD34+细胞、CD34+CD38-及粒/单系集落形成单位(CFU-GM)、红系爆式集落形成单位(BFU—E)均存在显著相关性(P<0.05),而与采集的单个核细胞(MNC)总数、CD34+Thy1+、AC133+细胞含量间无相关关     

Allogeneic and autologous stem-cell transplantation in advanced Ewing tumors

Background:An update of results from the High Risk Protocol ofthe Meta-EICESS Study, conducted at the Pediatric Stem-Cell Transplant Centersof Düsseldorf and Vienna. In order to evaluate a possible therapeuticbenefit after allogeneic SCT in patients with advanced Ewing tumors (AET), wecompared outcome after autologous and allogeneic stem-cell transplantation(SCT). Patients and methods:We analyzed 36 patients treated with themyeloablative Hyper-ME protocol (hyperfractionated total body irradiation,melphalan, etoposide ± carboplatin) between November 1986 and December1994. Minimal follow-up for all patients was five years. All patientsunderwent remission induction chemotherapy and local treatment beforemyeloablative therapy. Seventeen of thirty-six patients had multifocal primaryEwing's tumor, eighteen of thirty-six had early, multiple or multifocalrelapse, one of thirty-six patients had unifocal late relapse. Twenty-six ofthirty-six were treated with autologous and ten of thirty-six with allogeneichematopoetic stem cells. We analyzed the following risk factors, that couldpossibly influence the event-free survival (EFS): number of involved bones,degree of remission at time of SCT, type of graft, indication for SCT, bonemarrow infiltration, bone with concomitant lung disease, age at time ofdiagnosis, pelvic involvement, involved compartment radiation,histopathological diagnosis. Results:EFS for the 36 patients was 0.24 (0.21) ± 0.07.Eighteen of thirty-six patients suffered relapse or died of disease, nine ofthirty-six died of treatment related toxicity (DOC). Nine of thirty-sixpatients are alive in CR. Age 17 years at initial diagnosis (P< 0.005) significantly deteriorated outcome. According to the type ofgraft, EFS was 0.25 ± 0.08 after autologous and 0.20 ± 0.13after allogeneic SCT. Incidence of DOC was more than twice as high afterallogeneic (40%) compared to autologous (19%) SCT, even thoughthe difference did not reach significance (P = 0.08, Fisher's exacttest). Conclusions:Because of the rather short observation period,secondary malignant neoplasm (SMN) may complicate the future clinical courseof some of our patients who are currently viewed as event-free survivors. EFSin AET is not improved by allogeneic SCT due to a higher complication rate.The patient group was to small to analyze for a possiblegraft-versus-tumor effect.     

Selection and immunomagnetic purging of peripheral blood CD34+ cells for autologous transplantation in B-cell non-Hodgkin's lymphomas

Background: Clonogenic tumor cells in the hematopoietic progenitor cell harvest may contribute to relapse after high dose therapy for B-cell malignancies. Purging of the HPC harvest requires large amounts of anti-B-cell antibodies, whereas CD34-selection enriches self renewing HPC's but malignant cells are still detectable in many CD34+ fractions.Patients and methods: We examined the feasability and safety of a CD34-selection followed by purging with anti-B-cell antibodies in 11 patients with B-cell non-Hodgkin's lymphomas undergoing high-dose therapy with cyclophosphamide, BCNU and etoposide with retransfusion of autologous HPC's.Results: A mean number of 340 × 10⁸ mononuclear cells was used for CD34-selection and immunomagnetic purging. CD34+ cells were enriched from a mean of 1.7% (range 0.2%–4.5%) to a mean of 68% (range 49%–87%) with a mean recovery of 27% (range 15%–43%). The mean number of retransfused CD34+ cells was 1.2 × 10⁶/kg (range 0.6–2.2 × 10⁶/kg) body weight with a median of 11 days (range 10–13 days) to neutrophil recovery of 0.5 × 10⁹/l and 17 days (range 13–25 days) to platelet recovery of 50 × 10⁹/l. Mean number of intravenous antibiotics and inpatient days were 8 (range 0–14) and 22 (range 19–26) respectively. Major toxicity consisted in four septicemias.Conclusions: CD34-selected and purged HPC's are safe and mediate rapid hematological recovery after high dose therapy for B-cell non-Hodgkin's lymphomas.     

超高CD34+采集的动员方案序贯二次自体造血干细胞移植治疗难治性 霍奇金淋巴瘤

目的:探讨超高CD34+采集的动员方案后序贯二次自体造血干细胞移植治疗难治性霍奇金淋巴瘤的疗效和安全性。方法:对1例经过多疗程一线、二线、新药、免疫等均难治的霍奇金淋巴瘤患者,予以IA+C方案化疗+G-CSF动员干细胞后采集出超高水平CD34+细胞,之后行自体造血干细胞移植,移植后获得完全缓解,再予序贯第二次自体造血干细胞移植进行巩固治疗。结果:总计输注单个核细胞数13.67×108/kg,CD34+细胞48.68×106/kg,第一次自体造血干细胞移植术后第7天造血功能恢复,复查全身PET-CT提示获得完全缓解,第二次自体造血干细胞移植术后第8天造血功能恢复,两次自体造血干细胞移植相关并发症均在可控范围内。结论:超高CD34+细胞采集的IA+C方案化疗+G-CSF动员可以让患者有机会进行多次自体造血干细胞移植,是临床动员的创新方案。对于难治性霍奇金淋巴瘤,序贯二次自体造血干细胞移植可达到更深层次缓解,且安全性较高,延长患者无疾病生存期及总生存期,为难治性霍奇金淋巴瘤治疗提供更多临床依据。