高三尖杉酯碱对慢性粒细胞白血病患者骨髓CD+34细胞端粒酶活性的影响

目的:观察高三尖杉酯碱(HHT)对慢性粒细胞性白血病(CGL)患者骨髓CD+34细胞端粒酶活性的影响.方法:采用TRAP端粒酶测定法测定与HHT短期培养后的患者骨髓CD+34细胞端粒酶活性的变化.结果:培养24 h后,HHT使CGL患者骨髓CD+34细胞端粒酶活性下降了51%,而正常CD+34细胞端粒酶活性仅下降了32%,差异有显著性(P＜0.05).羟基脲(HU)使CGL患者骨髓CD+34细胞端粒酶活性下降了21%,使对照组酶活性下降了24%,差异无显著性(P＞0.05).结论:HHT对CGL患者和正常骨髓CD+34细胞端粒酶活性均有抑制作用,其抑制作用强于HU;HHT对CGL患者CD+34细胞端粒酶活性的抑制作用强于对正常CD+34细胞的抑制.     

慢性粒细胞白血病CD+34细胞的生物学特性

慢性粒细胞白血病(CML)是一种起源于骨髓多能造血干细胞的恶性克隆性疾病,其中90%以上的病例具有ph染色体,其分子基础是bcr/abl基因重排.CD34抗原是一种表达于造血干/祖细胞的膜表面糖蛋白,正常CD+34细胞亚群中90%以上为祖细胞和干细胞,骨髓中约1.5%的单个核细胞表达CD34抗原,未经刺激的外周血中0.1%～0.2%的单个核细胞为CD+34,体内单独或联合应用造血生长因子能够提高外周血中CD+34细胞的数量,并有效地应用于外周血干细胞移植(PBSCT).     

Expression of Caspase－3 in Cord Blood CD34^＋ Cells during Culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34^ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34^ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34^ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34^ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34^ cells during expansion in vitro.     

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Clini MACS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64%,纯度为98.2%;移植后白细胞恢复至》1.0×10 9/L和血小板》20×10 9/L的天数(中位数)分别为14 d和13 d.患者总体生存率66.7%(10/15),无疾病生存率53.3%(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.     

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.     

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。     

高三尖杉酯碱联合干扰素治疗慢性粒细胞白血病疗效分析

ph+慢性粒细胞白血病(CML)目前无条件进行异基因骨髓移植者,治疗大多仅为完全血液学缓解(CHR),个别有细胞遗传学反应,如何提高CML的疗效,延长缓解期仍然是治疗的难点.我科从2000年3月以来,开始应用高三尖杉酯碱(HHT)联合干扰素(IFN-α2b),统计结果显示,二药联合使用有良好的细胞遗传学反应,毒副作用轻微,现报告如下.     

The Influence of IFN-α on Blood Plasmacytoid Dendritic Cell in Chronic Myeloid Leukaemia

OBJECTIVE To study the mechanism of IFN on CML.METHODS Samples of 15 CML patients and 10 healthy controls were studied. The flow cytometry was performed to identify circulating pDCs. The concentration of IFN-α in serum and that in the supematant of peripheral blood mononuclear cells (PBMCs)cultured after stimulation with CpG ODN2216 were examined both in CML patients and in the healthy controls RESULTS There was significant reduction in the number of circulating pDCs, serum concentration of IFN-α and the capacity of IFN-α producing PBMCs in CML patients compared with those in healthy control individuals (P < 0.001). After the active treatment with IFN-α and hydroxyurea, the quantity and function of pDCs were increased in stabilized patients, especially the function of pDCs in 2 patients achieving major cytogenetic.response (MCR). The proportion and function of pDCs and the serum levels of IFN were inversely correlated with both WBC and age of the patients with CML, and positively correlated with the state of the illness.dysfunction of circulating pDCs. The active treatment with IFN in CML patients may be related to the restoration of pDCs.     

Analysis of the Clonal Expansion of TCR VβT Cells in Patients

Objective To investigate the clonal expansion of T cell receptor(TCR)Vβ subfamily T cells which were considered as GVL effective cells after donor lymphocytes infusion(DLI)in patients with relapse chronic myelogenous leukemia(CML)after allogeneic bone marrow transplantation(allo-BMT).Methods The CDR3 of TCR Vβ24 subfamily genes were amplified in samples of peripheral blood mononuclear cells at different time points before and after DLI,which were drawn from 2 cases of relapse CML treated by allo-BMT,to observe the usage of TCR Vβrepertoire.The PCR products were further labeled with fluorescent and analyzed by genescan technique for identification of the CDR3 size,to evaluate the clonality of the detectable TCR VβT cells.Results Only 4-11 VβT subfamily T cells could be identified in CML cases before DLI,and 12-21 Vβ subfamily T cells could be deected in samples from CML which display remission after DLI.Genescan analysis showed that new clonal expansion TCR Vβ subfamily T cells could be found in samples after DLI.Conclusion The skew distribution of TCR Vβ subfamily T cells could be found on patients with relapse CML after allo-BMT,and this skewing pattern may stage to stage to normal pattern during the complete remission.The GVL effect may exert through some clonal expansion TCR Vβ subfamily T cells during the treatment of DLI in relapse CML.     

Long-term effect of homoharringtonine on chronic granulocytic leukemia

From 1996, low-dosage (1.5 mg/m2) homo- harringtonine (HHT) was used to treat chronic granulocytic leukemia in chronic phase (CGL-CP) patients for remission induction and long term maintenance treatment. It was found that the period of chronic phase intervals was longer in low-dosage HHT group than that in hydroxyurea (HU) group and HHT could induce apoptosis of bone marrow CD34+ cells from CGL-CP patients. MATERIALS AND METHODS Patients Newly diagnosed CGL-CP patients …     

Expression of caspase-3 in cord blood CD34<Superscript>+</Superscript> cells during culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34⁺ cells from cord blood (CB) during culturein vitro with different growth factors.Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34⁺ CB cells during culturein vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34⁺ cells. The expression of caspase-3 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34⁺ cells as well as during the first 3 days expansion with cytokines. With longer culture timein vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34⁺ cells during expansionin vitro. The study was supported by a grant from the National Natural Science Foundation of China (No. 39928010)     

Promyelocytic blast crisis in chronic granulocytic leukemia with 15;17 translocation

A patient with Philadelphia chromosome Ph¹-positive chronic granulocytic leukemia (CGL) developed an acute transformation associated with a hemorrhagic diathesis and bone marrow infiltration by hypergranular promyelocytes and blasts. Cytogenetic analysis of bone marrow metaphase cells at transformation showed a t(15;17) superimposed upon the Ph¹-positive cell line. These findings suggest a critical relationship between the t(15;17) and arrest of myeloid leukemic cell differentiation at the promyelocyte stage.     

Evaluation of residual CD34+Ph+ progenitor cells in chronic myeloid leukemia patients who have complete cytogenetic response during first‐line nilotinib therapy

BACKGROUND:

Compared with imatinib, nilotinib is a potent breakpoint cluster region/v‐abl Abelson murine leukemia viral oncogene (bcr‐abl) kinase inhibitor, and it induces higher rate and rapid complete cytogenetic response (CCyR), yet no clinical data are available regarding its efficacy against chronic myeloid leukemia (CML) stem cells. Earlier studies demonstrated that clusters of differentiation 34–positive, Philadelphia chromosome–positive (CD34⁺Ph⁺) cells are detectable in about 45% of patients with CML, despite being on long‐term imatinib therapy and having achieved sustained CCyR.

METHODS:

CD34⁺ cells from bone marrow of de novo CML patients in the chronic phase (n = 24) treated with nilotinib (median duration of therapy, 22 months) were isolated and scored for BCR‐ABL by fluorescent in situ hybridization (FISH) analysis. Similar analysis was also performed in 5 de novo CML chronic phase patients who achieved CCyR within 3 months of nilotinib therapy.

RESULTS:

FISH evaluation of a median of 100 CD34⁺ nuclei per patient revealed that only 1 of 20 (5%) evaluable patients showed residual Ph⁺ progenitor cells. In this patient, just 1 of 140 (0.7%) CD34⁺ interphase nuclei was found to be positive for BCR‐ABL. Surprisingly, no CD34⁺Ph⁺ cells were found even in those 5 patients evaluated after 3 months of nilotinib treatment.

CONCLUSIONS:

This study assessed for the first time the persistence of CD34⁺Ph⁺ cells during nilotinib first‐line treatment. Preliminary results showed that in patients in CCyR, even after short‐term nilotinib therapy, residual leukemic progenitors are very rarely detected compared with imatinib‐treated CCyR patients. It is yet to be determined if these findings will have an impact in the path to a cure of CML with tyrosine kinase inhibitors. Cancer 2012. © 2012 American Cancer Society.     

高三尖杉酯碱对慢性髓细胞白血病患者T细胞亚群和Th细胞亚群的影响

 目的 研究慢性髓细胞性白血病（CML）患者高三尖杉酯碱（HHT）治疗前后细胞免疫功能的变化。方法 测定CML患者初诊时及HHT治疗后T细胞亚群和Th细胞亚群的变化并观察初诊患者外周血单个核细胞（PBMNC）在体外培养过程中Th细胞亚群的变化。结果 初诊时患者T细胞中以CD+4 T细胞占优势,Th亚群中以Th2细胞占优势,HHT治疗后达主要细胞遗传学反应患者则分别转变成以CD+8 T细胞和Th1细胞占优势。初诊患者PBMNC在培养前和培养早期以Th2细胞占优势,培养24 h和48 h则Th1细胞占优势。结论 CML患者存在免疫监视功能低下,HHT治疗获主要细胞遗传学反应患者的细胞免疫功能则明显恢复。     

急性髓细胞白血病细胞CD34抗原表达的临床及生物学意义

为研究ＣＤ３４在急性髓细胞白血病（ＡＭＬ）中的临床及生物学意义，用流式细胞术检测了１０７例ＡＭＬＣＤ３４表达。结果显示４８．５％的患者ＣＤ３４阳性表达，Ｍ３（１５．８％）低于其他亚型（ｐ〈０．０５）。ＣＤ３４＾＋者肝脾肿大常见。ＣＤ３４表达在ｔ（８；２１）、正常核型及ｔ（１５；１７）组分别为７２．０％、４０?７％及１７．６％。ＣＤ３４＾＋者的完全缓解率（４１．９％）明显低于ＣＤ３４＾－者的５９．６     

Isolation and transplantation of highly purified autologous peripheral CD34<Superscript>+</Superscript> progenitor cells: purging efficacy,hematopoietic reconstitution following high dose chemotherapy in patients with breast cancer: results of a feasibility study in Japan

BACKGROUND: High-dose chemotherapy with autologous stem cell support may have some therapeutic impact on certain groups of the patients with advanced breast cancer(BRCA). Since stem cell contamination by tumor cells might contribute to relapse, development of a tumor cell purging technique would improve the clinical outcome. The present study was undertaken to evaluate the purging efficacy of autologous mobilized CD34+peripheral stem cells in patients with breast cancer (BRCA) in an advanced stage or relapse. METHODS: CD34+cells were selected from autologous peripheral blood stem cells (PBSC) using a clinical scale of magnetic-activated cell sorting system (CliniMACS), followed by high-dose chemotherapy with transplantation of CD34+ selected cells. Amplification of cytokeratin 19 (CK19) and 20 (CK20) gene in leukapheresis products were measured to evaluate the performance of tumor cell elimination. RESULTS: Seven patients were entered into this study. After leukopheresis, 1 patient was dropped form this study due to poor mobilization. Among 6 patient, a total of 8 CD34+ selection was performed. The median purity and recovery rate of the CD34+ cells post selection was 85.1% (range 62.5-98.1%) and 74.2% (range 50.2-90.2%), respectively. After isolation of CD34+cells, the elimination rate in the logarithmic transformation of CK19 was 2.77 log, and that of CK20 were 2.43 log and 2.53 log. In 4 patients, high-dose chemotherapy was performed, followed by the transplantation of the isolated CD34+cells. Rapid neutrophil recovery, as well as platelet recovery was seen with a median time to reach 0.5 x 109/l neutrophils of 9 days(range 8-9), and 20 x 109/l platelets of 12 days (range 10-13). There was no treatment related death and no serious adverse events directly associated with the selection procedure or infusion of selected cells. CONCLUSIONS: The present study demonstrated that the CliniMACS system is a highly effective positive selection method and that a high purging efficacy could be obtained without compromising the hematopoietic reconstitution capacity of the graft in BRCA patients undergoing high-dose chemotherapy.     

CD15<Superscript>+</Superscript>/CD16<Superscript>low</Superscript> human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function

Myeloid-derived suppressor cells (MDSCs) are a subpopulation of myeloid cells with immunosuppressive function whose numbers are increased in conditions such as chronic infection, trauma, and cancer. Unlike murine MDSCs defined as CD11b⁺/Gr-1⁺, there are no specific markers for human MDSCs. The goal of this study was to delineate a specific human MDSCs subpopulation in granulocytes from terminal cancer patients and investigate its clinical implications. Here, we show that the CD15⁺/CD16^low subset was increased in terminal cancer patients compared with healthy donors (P = 0.009). Phorbol 12-myristate 13-acetate-activated granulocytes (CD16^low/CD66b⁺⁺/CD15⁺) that have a phenotype similar to MDSCs from cancer patients, effectively suppressed both proliferation and cytotoxicity of normal T cells. Among cancer patients, T-cell proliferation was highly suppressed by granulocytes isolated from terminal cancer patients with a high proportion of CD15⁺/CD16^low cells. Patients with low peripheral blood levels of CD15⁺/CD16^low cells had significantly longer survival than those with high levels (P = 0.0011). Patients with higher levels of CD15⁺/CD16^low also tended to have poor performance status (P = 0.05). These data suggest that CD15⁺/CD16^low granulocytes found in terminal cancer patients may play a role in the progression of cancer by inhibiting tumor immunity.