EFFECT OF SHORT-COURSE, HIGH-DOSE STEROID THERAPY IN A CHILD WITH MYELODYSPLASTIC SYNDROME

High-dose methylprednisolone (HDMP) has been shown to induce differentiation of myeloid leukemic cells with a remarkable antileukemic effect in children with various subtypes of acute myeloblastic leukemia (AML). Here the beneficial effect of short-course HDMP therapy in a child with myelodysplastic syndrome (MDS) is reported. Oral methylprednisolone sodium succinate (Prednol-L) was administered at a single daily dose of 30 mg/kg for 5 days to a 4-year-old girl with refractory anemia with excess of blasts and hypocellular bone marrow before the initiation of chemotherapy. In addition to dramatic clinical improvement, the patient's white blood cell count increased from 2.3 ×10 9 /L to 5.0 ×10 9 /L, and peripheral blood blast cells disappeared 4 days after HDMP treatment. Repeated bone marrow aspirate 1 week after the initiation of HDMP disclosed increased cellularity with no blasts. Furthermore, short-course HDMP treatment stimulated the increase in the number of peripheral blood lymphocytes and CD3 + , CD4 + , CD8 + , CD19 + , CD34 + , and NK cells. Results obtained with HDMP from the previous studies and the present case suggest that high-dose methylprednisoloneis a promising agent in the treatment of MDS and it is recommended as an initial treatment especially for MDS children with hypocellular bone marrow at presentation.     

EFFECT OF SHORT-COURSE,HIGH-DOSE STEROID THERAPY IN A CHILD WITH MYELODYSPLASTIC SYNDROME

High-dose methylprednisolone (HDMP) has been shown to induce differentiation of myeloid leukemic cells with a remarkable antileukemic effect in children with various subtypes of acute myeloblastic leukemia (AML). Here the beneficial effect of short-course HDMP therapy in a child with myelodysplastic syndrome (MDS) is reported. Oral methylprednisolone sodium succinate (Prednol-L) was administered at a single daily dose of 30 mg/kg for 5 days to a 4-year-old girl with refractory anemia with excess of blasts and hypocellular bone marrow before the initiation of chemotherapy. In addition to dramatic clinical improvement, the patient's white blood cell count increased from 2.3 &#50 10 9 /L to 5.0 &#50 10 9 /L, and peripheral blood blast cells disappeared 4 days after HDMP treatment. Repeated bone marrow aspirate 1 week after the initiation of HDMP disclosed increased cellularity with no blasts. Furthermore, short-course HDMP treatment stimulated the increase in the number of peripheral blood lymphocytes and CD3 + , CD4 + , CD8 + , CD19 + , CD34 + , and NK cells. Results obtained with HDMP from the previous studies and the present case suggest that high-dose methylprednisoloneis a promising agent in the treatment of MDS and it is recommended as an initial treatment especially for MDS children with hypocellular bone marrow at presentation.     

A NOVEL APPROACH TO TREATMENT WITH HIGH-DOSE STEROID AS A DIFFERENTIATION INDUCER IN CHILDREN WITH ACUTE MYELOBLASTIC LEUKEMIA

Several experimental studies have demonstrated that certain steroid hormones can induce differentiation of mouse myeloid leukemic cells to macrophages and granulocytes. We have shown that high-dose methylprednisolone treatment (HDMP, 20-30 mg/kg/day) can induce differentiation of leukemic cells to mature granulocytes in children with different morphological subtypes of acute myeloblastic leukemia (FAB AML M1, M2, M3, M4). In addition, apoptosis can also be induced in vivo and in vitro in AML blast by HDMP treatment. Short-course (3 to 5 days) HDMP treatment increases the hematopoietic CD34- positive progenitor cells in both bone marrow and peripheral blood in children with AML. Acceleration of leukocyte and neutrophil recovery has been obtained by the administration of short-course HDMP in chemotherapy-induced neutropenic children with AML. The addition of HDMP to anti leukemic chemotherapy increased the complete remission rate and prolonged the duration of remission in children with AML and significantly improved the outcome of AML children who presented with extramedullary infiltration. We suggest that the possibility of HDMP-induced differentiation and apoptosis should be evaluated in patients with other malignant diseases.     

CD11b对儿童急性白血病脐血移植粒细胞和血小板恢复时间的影响

为了研究CD34+ CD1 1b+ 细胞输入量在脐血移植中对中性粒细胞和血小板恢复时间的影响 ,观察并分析 1 7例儿童急性白血病患者进行无关脐血移植的临床数据。结果显示 ,移植后 1 7例患者中性粒细胞≥ 0 5× 1 0 9 L的时间为 1 1～ 32天 (中位数为 1 7天 ) ;血小板≥ 2 0× 1 0 9 L的时间为 1 2～ 1 1 8天 (中位数为 4 0天 )。CD34+CD1 1b+ 细胞输入量为 1 0 7～ 79 0 0× 1 0 4 Kg(中位数为 9 1 8× 1 0 4 Kg)。CD34+CD1 1b+ 细胞输入量与中性粒细胞恢复时间呈反比 (γ =- 0 4 89,P <0 0 5) ;CD34+CD1 1b+ 细胞输入量与血小板恢复时间无关 (r =- 0 2 38,P >0 0 5)。CD34+ CD1 1b+细胞输入量与造血干细胞移植时的造血重建有关。     

Comparision of the apoptotic effects on lymphoblasts and on increase of myeloid lineage cells of a short-time, high-dose methylprednisolone and the conventional-dose prednisolone treatments in children with acute lymphoblastic leukemia

The authors compare the apoptotic effect on the lymphoblasts and the proliferative effect on the myeloid lineage cells of a short-course high-dose methylprednisolone (HDMP) and the conventional-dose prednisolone treatments in children with acute lymphoblastic leukemia (ALL). The patients were divided into 2 groups. Group I (n = 10) received HDMP (30 mg/kg/day for 7 days) in a single dose before 6 a.m. perorally. Group II (n = 10) received prednisolone (2 mg/kg/day for 7 days) in 3 doses. The apoptotic percentages of lymphpblasts and the percentages of blasts and myeloid lineage cells were determined after performing the bone marrow aspiration (BMA) at diagnosis on the 0th, 3rd, and 7th days of the treatments in all patients. The mean apoptotic percentages of the lymphoblasts on the 3rd day were significantly higher than those on the 0th and 7th days in both groups (p < .05). The highest apoptosis was determined on the 3rd day in group I. The mean percentages of the blast cells on the 7th day were significantly lower than those on the 0th and the 3rd days in both groups (p < .05). The lowest lymphoblast percentage was determined on the 7th day in group I. The mean percentages of the CD13+ and CD33+ cells on the 7th day were significantly higher than those on the 0th and the 3rd days in both groups (p < .05). The highest percentages of the CD13+ and CD33+ cells were found on the 7th day in group I. Prednisolone and HDMP showed no proliferative effect on the CD14+ cells. These findings indicate that a short-course HDMP treatment shows a more effective apoptosis on the lymphoblasts and on the increase of the myeloid lineage cells when compared to the prednisolone treatment. The authors suggest that HDMP may be used in the treatment of patients with ALL instead of prednisolone.     

Proliferation of myeloid lineage cells and apoptosis of lymphoblastic leukemic cells induced by short-course high-dose methylprednisolone in patients with acute lymphoblastic leukemia

In this paper, we investigated the effects of short-course high-dose methylprednisolone (HDMP) treatment on the proliferation of myeloid lineage cells and on apoptosis of blast cells in eight children with acute lymphoblastic leukemia (ALL). The patients were given the HDMP treatment (30 mg/kg/d, perorally) before 9:00 a.m. for seven days. Bone marrow (BM) aspiration was done at days 0 and 3 of the HDMP treatment in all patients and at the 7th day of the HDMP treatment in six patients. Bone marrow blast cells had gradually decreased after the HDMP treatment by the 7th day. There were statistically significant differences between the mean percentages of BM blast cells at days 0 and 3, days 0 and 7, and at days 3 and 7 (p<0.05). The mean percentages of blast cell apoptosis at the 3rd day was significantly higher than at days 0 and 7, and apoptosis at day 0 was significantly lower than at the 7th day (p<0.05). The mean percentages of BM myeloid lineage cells at the 7th day was significantly higher than at days 0 and 3 (p<0.05), and the mean percentage at day 0 was significantly lower than at the 3rd day (p<0.05). These findings indicate that short-course HDMP treatment causes apoptosis on lymphoblasts and increases the proliferation of myeloid lineage cells in children with ALL.     

The role of short course of high-dose methylprednisolone in children with acute myeloblastic leukemia (FAB M2) presented with myeloid tumor

The authors have previously demonstrated a favorable effect of high-dose methylprednisolone (HDMP), which can induce differentiation and apoptosis of leukemic cells in children with acute myeloblastic leukemia (AML). Here, they evaluate the effect of short-course HDMP in 2 children with acute myeloblastic leukemia (AML-M2) presented with myeloid tumor (MT). Methylprednisolone (20 or 30 mg/kg/day) was given orally, in a single dose, without using other antileukemic agents. Rapid cytoreduction in MT, peripheral blood, and bone marrow blasts was observed in both children following short-course (4 or 7 days) HDMP treatment, possibly due to HDMP-induced differentiation and apoptosis of leukemic cells. The effects of HDMP should be explored in patients with other subtypes of AML who present with MT.     

TREATMENT OF KASABACH-MERRITT SYNDROME BY MEGADOSE METHYLPREDNISOLONE

The authors have previously demonstrated a favorable effect of high-dose methylprednisolone (HDMP), which can induce differentiation and apoptosis of leukemic cells in children with acute myeloblastic leukemia (AML). Here, they evaluate the effect of short-course HDMP in 2 children with acute myeloblastic leukemia (AML-M2) presented with myeloid tumor (MT). Methylprednisolone (20 or 30 mg/kg/day) was given orally, in a single dose, without using other antileukemic agents. Rapid cytoreduction in MT, peripheral blood, and bone marrow blasts was observed in both children following short-course (4 or 7 days) HDMP treatment, possibly due to HDMP-induced differentiation and apoptosis of leukemic cells. The effects of HDMP should be explored in patients with other subtypes of AML who present with MT.     

环磷酰胺对小鼠骨髓造血干/祖细胞作用及机制研究

目的探讨环磷酰胺(CY)对小鼠骨髓造血干/祖细胞损伤的作用特点与机制。方法分别以大(380mg/kg)、中(200mg/kg)、小(100mg/kg)三种剂量CY腹腔注射BALB/C小鼠,注射后一周内动态检测外周血白细胞(WBC)、骨髓及外周血CD34+细胞含量、骨髓有核细胞数(NC)与细胞凋亡及病理学改变。结果①在CY处理后3～4d内,骨髓病理损伤逐渐加重,外周血WBC和骨髓NC逐渐下降直至最低值,受抑程度与CY剂量呈显著正相关。②外周血CD34+细胞与骨髓CD34+细胞水平变化基本一致,均在CY注射后1～3d进行性降低,随后中、小剂量组先一过性迅速上升至高出正常水平,然后再下降,而大剂量组则一直处于缓慢回升。③CY处理后1～3d内骨髓细胞凋亡明显增加。结论CY短期内对小鼠造血干/祖细胞损伤存在剂量—时间效应关系,中、小剂量起“动员”作用,大剂量起“摧毁”作用,诱导细胞凋亡是造血损伤的机制之一。     

卡介苗体外对急性淋巴细胞性白血病患儿细胞毒性T淋巴细胞杀伤HL-60细胞效应的影响

目的：探讨卡介苗（BCG）体外对急性淋巴细胞性白血病(ALL)患儿外周血细胞毒性T淋巴细胞（CTL）杀伤HL-60细胞效应的影响。方法：应用Ficoll-Hypaque法分离ALL患儿（白血病组）和健康儿童（对照组）的外周血单个核细胞,在白介素-2（IL-2）、植物血凝素（PHA）和BCG作用下诱导成CTL细胞;应用流式细胞仪测定CD3、CD3+CD4+、CD3+CD8+比例变化;应用MTT法检测CTL对HL-60细胞的杀伤力。结果：培养前2 d,对照组和白血病组CTL细胞数量及体积均无明显变化,第3天开始两组细胞均开始增殖,体积变大,6～10 d达高峰;培养至第10天时,加入BCG的白血病组和对照组细胞数量分别较无BCG的白血病组和对照组增多;白血病组CD3+CD8+比例明显高于对照组,经BCG作用后两组CD3+CD8+比例均明显升高;白血病组CTL对HL-60细胞的杀伤力明显低于对照组。结论：BCG可协同IL-2、PHA体外促进CTL细胞增殖并提高其对HL-60细胞的杀伤力。     

急性白血病患儿CD4~+CD25~+调节性T细胞和NK细胞免疫作用探讨

目的观察外周血CD4+CD25+调节性T细胞(CD4+CD25+Treg)及自然杀伤细胞(na-ture killer cell,NK)在白血病患儿及非白血病患儿的不同,了解白血病患儿的免疫状态,探讨CD4+CD25+Treg细胞及NK细胞在小儿急性白血病肿瘤免疫中的意义。方法以流式细胞术检测急性白血病初诊患儿及非白血病患儿各30例的外周血CD4+CD25+Treg细胞、NK细胞的数量及比例。结果外周血CD4+CD25+CD127-细胞占CD4+T细胞的比例白血病组为(11.45±1.41)%,显著高于对照组为(6.98±1.09)%(P<0.05)。而NK细胞数量白血病组为(5.13±2.97)%,显著低于对照组为(15.06±3.91)%(P<0.05)。结论 (1)急性白血病患儿外周血CD4+CD25+Treg细胞数量升高,NK细胞数量降低,表明急性白血病患儿NK细胞免疫功能处于抑制状态。CD4+CD25+Treg细胞可能在白血病的发生、发展中起一定作用。(2)通过检测CD4+CD25+CD127-T细胞可较好的反映CD4+CD25+Treg细胞的比例,简便可行、重复性好、检测结果准确、可靠。     

Granulocyte colony-stimulating factor alone at 20 micrograms/kg vs. 10 micrograms/kg for peripheral blood stem cell mobilization in children

Mobilization of peripheral blood stem cells (PBSC) by granulocyte colony-stimulating factor (G-CSF), at 10 micrograms/kg/day vs. 20 micrograms/kg/day (in 42 and 29 patients, respectively), was compared in children with solid tumors or leukemias. During mobilization, differences were noted in the peak values of CD34+ cells in peripheral blood (PB) in these two groups (median 28 x 10(6)/L for 10 micrograms/kg/day vs. 61 x 10(6)/L for 20 micrograms/kg/day; p = 0.025). Similar numbers of progenitor cells were harvested for the two concentrations of G-CSF. However, similar CD34+ cell levels in the leukapheresis product were obtained after only the third dose of G-CSF at 20 micrograms/kg/day compared with the fourth dose of G-CSF at 10 micrograms/kg/day (1.7 and 1.2 x 10(6) CD34+ cells/kg/one patient's blood volume processed, respectively). Of note is the impact of diagnosis on PB CD34+ cell levels. We conclude that, in children, mobilization with G-CSF at 20 micrograms/kg/day could minimize the duration of priming but not reduce the number of leukaphereses. Thus, the impact on outcome, clinical practice, bed utilization, and health economics is uncertain.     

CD+34CD+62L细胞输入量对儿童急性白血病无关脐血移植的影响

目的　研究CD 3 4 CD 62L细胞输入量在无关脐血移植治疗儿童急性白血病中对造血干细胞植入、中性粒细胞和血小板恢复时间的影响。方法　用流式细胞术分析复苏后的CD 3 4 CD 62L细胞数 ,并对 2 3例急性白血病儿童在无关脐血移植后的中性粒细胞和血小板恢复时间等临床资料进行测定。结果　 2 3例患儿中 ,2 1例被植入 ,其中性粒细胞 >5 0 0 / μl的时间为 11～ 2 3d(中位数为 17 5d) ,而在 18例患儿中 ,血小板 >2万 / μl的时间为 12～ 118d(中位数 4 1d)。当CD 3 4 CD 62L细胞输入量 >1 3× 10 5/kg时 ,有利于造血干细胞的植入 ,中性粒细胞的恢复时间与CD 3 4 CD 62L细胞输入量存在一定的相关性趋势 ,γ值为 - 0 32 4 ,0 0 5

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TPO和FL有效扩增脐血CD34+细胞为巨核前体细胞

目的探讨人脐血CD34+细胞在TPO和FL细胞因子组合作用下体外扩增为巨核前体细胞的效果,为开展血小板减少症的细胞治疗建立实验条件。方法本研究采用人脐血经免疫磁珠法纯化分离的CD34+细胞,研究TPO+FL细胞因子组合对体外巨核前体细胞的扩增效果。结果显示在TPO与FL组合的作用下,脐血CD34+细胞(n=6)显著地扩增为巨核前体细胞(CD61+CD41+细胞),作用14天为较理想时间。无论是全细胞数和CD34+细胞,或是CD61+CD41+细胞和CFUMK,经过体外扩增,均显著增加。结论研究表明脐血CD34+细胞在TPO和FL细胞因子组合作用下可有效地扩增为巨核前体细胞,第14天是培养较理想时间。     

抗CD47抗体联合阿糖胞苷靶向治疗NOD/SCID小鼠单核细胞白血病的研究

目的：探讨CD47在急性髓细胞白血病NOD/SCID小鼠模型中的预后意义及靶向治疗的最佳策略。方法：将分选的CD34+CD38-白血病干细胞（leukemia stem cells, LSCs）移植入NOD/SCID小鼠体内,建立小鼠急性单核细胞白血病模型;抗人CD47单克隆抗体单独或联合阿糖胞苷治疗白血病小鼠7~14 d,并进行疗效分析。将LSCs与小鼠巨噬细胞在含抗CD47单克隆抗体的培养液中共培养,观察CD47对巨噬细胞吞噬LSCs能力的影响。结果：THP-1细胞中存在CD34+CD38- LSCs,比例约为0.12%±0.06%,将分选后的CD34+CD38- LSCs（比例高达97.0%±1.7%）移植入NOD/SCID小鼠后成功建立白血病模型。体内实验表明,阿糖胞苷（7 d）联合抗CD47单克隆抗体（14 d）治疗后,白血病小鼠外周血和骨髓中CD33+CD45+白血病细胞下降最明显（P<0.01）,生存时间明显长于其它各组。体外共培养2 h后,抗CD47单克隆抗体组吞噬指数（76.9%±12.2%）明显高于抗CD45单克隆抗体组（7.60%±2.4%,P<0.01）。结论：CD47高表达是急性髓细胞白血病的预后不良因素。阿糖胞苷联合抗CD47单克隆抗体可有效杀灭普通白血病细胞和白血病干细胞,对彻底治愈急性髓细胞白血病具有重要临床意义。     

DOUBLE HAPLOIDENTICAL TRANSPLANTATION OF HEMATOPOIETIC PROGENITOR CELLS IN A BOY WITH MYELODYSPLASTIC SYNDROME

A 12-year-old boy with myelodysplastic syndrome underwent a double transplantation of hematopoietic progenitor cells from his haploidentical brother. After conditioning with busulfan, cyclophosphamide, and Vepesid, the first bone marrow transplantation was performed using 3.53 10 6/kg of CD34+ cells. Initial engraftment was followed by graft rejection. The second conditioning consisted of melphalan and anti-thymocyte globulin. The boy was then transplanted with 5.15 10 6/kg of CD34+ cells, harvested from bone marrow (BM) and peripheral blood. Graft versus host disease (GvHD) prophylaxis consisted of cyclosporine A+ short methotrexate. Hematological recovery was rapid and stable. Acute GvHD I (skin) resolved after 2 weeks of steroid treatment. A relapse occurred on day + 140. At that time NK cells decreased from 20 to 7% with the lowest CD4+/CD8+ratio, 0.07. Just after relapse, the percentage of cytokine-induced killer cells (CIK-CD3+CD56+)dropped from 3.34 to 0.1%. CsA treatment was stopped and the patient received T cell (CD3+cells)add-back four times on days +146, + 199, + 234, and + 262 in doses of 0.5 10 5,1.0 105,2.0 105, and 4.0 105/kg, respectively. No acute GvHD occurred. Additionally, bone marrow biopsy before the second add-back showed complete remission. Analysis of lymphocyte subsets before the fourth add-back showed the highest values of CD4+, NK, and CIK cells and also the highest CD4 + /CD8 + ratio.     

Mobilization of peripheral blood progenitor cells using 16 versus 10 mg/kg/d G-CSF in children with malignancies

Administration of hematopoietic growth factors, with or without chemotherapy, can augment progenitor cell numbers available for collection. The dose of granulocyte colony stimulating factor (G-CSF) used for mobilization of peripheral blood progenitor cells (PBPC) is controversial, and doses between 5 and 32 microg/kg/d have been reported in adults. In order to determine the dose-response effect for G-CSF in mobilizing PBPC in children, we randomized 30 children with malignancies to receive either 16 or 10 microg/kg/d subcutaneously starting on the day after the disease-oriented chemotherapy regimen and continuing until the completion of leukapheresis. Leukapheresis commenced after threshold WBC > 1 x 10(9)/L was achieved and continued until 10 x 10(6) CD34+ cells/kg were obtained or for 6 procedures. Both treatment groups achieved an adequate yield of CD34+ cells with an average of 4 leukapheresis procedures. The numbers of CD34+ cells/kg were 8.3 x 10(6) and 11.7 x 10(6) in patients receiving 16 and 10 microg/kg/d doses of G-CSF, respectively, or 2.1 x 10(6) and 3.7 x 10(6) cells/kg per leukapheresis. The levels of CD34+ cells in peripheral blood had a wide interindividual variation, and were not significantly different after 16 or 10 microg/kg doses of daily G-CSF. We conclude that there is no advantage to using 16 microg/kg/d of G-CSF post-chemotherapy for PBPC mobilization in children.     

Expression of AC133 vs. CD34 in acute childhood leukemias

AC133, a newly discovered antigen on human progenitor cells, demonstrating 5-transmembranous domains is expressed by 30-60% out of all CD34+ cells. Our aim therefore was to investigate the extent of human stem-/progenitor cells expressing AC133 antigen in umbilical cord blood, peripheral blood without or following an application of granulocyte-colony stimulating factor (rhG-CSF). The main task was the investigation of bone marrow aspirates derived from children suffering from newly diagnosed acute leukemias, as well as from patients with a relapse or during a complete remission. The determination of antigen expression was done by application of flow cytometry (FACScan analysis) and the usage of newly developed monoclonal antibodies (AC133/1 and AC133/2; Miltenyi Biotec GmbH) in combination with monoclonal antibody directed against CD34-antigens (HPCA-2; BD). Our studies till now show average percentages in umbilical cord blood derived from 43 newborns about 0.294 +/- 0.165% AC133+ vs. 0.327 +/- 0.156% CD34+ hematopoietic stem-/progenitor cells (HSPC). In peripheral blood from 11 healthy donors we verified up to 0.15% CD34+ as well as AC133+ HSPC's. The concentration of progenitor cells was found to be obviously higher in peripheral blood from children with various diseases (neuroblastoma, rhabdomyosarcoma, ALL/AML) and undergoing application with rhG-CSF in order to be prepared for PBSC-transplantation. In those cases we found up to 3.51% AC133+ cells as well as slightly higher values (3.94%) for CD34 antigens. Additionally we quantified 128 bone marrow (BM) samples for AC133+ and CD34+ cells. In 10 BM samples, derived from patients without any neoplasia, the CD34+ cells were about 0.03% and 1.49%, whereas AC133 values were up to 0.64%. Bone marrow aspirates from 53 children with acute leukemias at time of diagnosis (ALL: n = 41/AML: n = 12) have been immunophenotyped and leukemic blast cells have been proved for AC133- and CD34 antigen expression. 32/41 (78%) of lymphoblastic leukemic cells showed to be positive for CD34 antigen and 24/41 (58%) demonstrated AC133 antigens. Interestingly there were 2 ALL-patients with pathological blast cells positive for AC133 but lacking of any CD34 antigens. 42% (5/12) of investigated AML patients showed CD34+ phenotype, on the other hand there were only 25% (3/12) with AC133+ phenotype. Similar values were found in relapsed patients (n = 18). In BM samples from patients during complete remission (n = 47) we could detect percentages up to 5.55% for CD34 and up to 1.25% for AC133 positive stem-/progenitor cells. Such quite high data may be explained by occasionally application of rhG-CSF therapy. Our results till now lead to the conclusion, that it seems to be useful, to recruit quantification of CD34+ HPSC by additionally detecting AC133 antigens. This new stem cell marker (AC133) may be of great value in case of autologous peripheral blood stem cell transplantation (PBSCT) because it could be an alternative to the usual CD34+ MACS selection system.     

脐血和外周血来源的巨核细胞体外扩增差异的研究

目的建立外周血(peripheral blood,PB)和脐血(cord blood,CB)来源的CD3+4细胞体外定向诱导扩增巨核细胞(megakaryocyte,MK)的最佳体系,探讨两种来源巨核细胞的扩增差异。方法以Ficoll-Hapaque分离“动员”的外周血和脐血单个核细胞,免疫磁珠分离纯化CD3+4细胞,在含胎牛血清的液体培养体系中,以不同细胞因子组合诱导两种来源的CD3+4细胞,定时进行细胞计数和流式细胞术检测培养体系中CD4+1细胞的含量;同时采用甲基纤维素半固体培养法进行巨核细胞集落培养,测定巨核细胞集落形成单位的数量。结果在血小板生成素(thrombopoietin,TPO)+胎肝酪氨酸激酶配体(FLT-3ligand,FL)+白介素6(interleukin-6,IL-6)+IL-3组合中,外周血来源的CD4+1细胞第10天扩增了131±18倍,脐血来源的CD4+1细胞在培养的第14天扩增了193±25倍,为增殖高峰。均明显高于同来源的其他3组(P<0.05),随着时间的推移两者的CD4+1细胞扩增倍数均呈下降趋势。结论TPO+FL+IL-6+IL-3组合均为CB和PB体外诱导扩增巨核细胞的最佳组合。CB来源的巨核细胞较PB来源的巨核细胞有更强的增生能力,而PB来源的CD3+4细胞产生巨核细胞的时间较CB来源的短,与临床上外周血造血干细胞移植的血小板造血重建快于脐血移植相一致。     

骨髓基质细胞及VLA-4抗体对儿童白血病细胞凋亡影响的研究

目的：研究骨髓基质细胞（BMSCs）对儿童白血病细胞的保护作用及细胞粘附分子缓慢抗原-4（VLA-4）抗体对原代白血病细胞凋亡的影响。方法：分离儿童白血病骨髓单个核细胞,培养BMSCs及白血病细胞。实验分4个组：①白血病细胞单独培养组（对照组）;②BMSCs＋白血病细胞组（BMSCs组）;③BMSCs上清+白血病细胞组（上清组）;④BMSCs＋白血病细胞＋VLA-4抗体组（抗体组）。流式细胞仪检测各组白血病细胞的凋亡率,RT-PCR方法检测各组白血病细胞survivin和bcl-2基因的表达。结果：BMSCs组和上清组的白血病细胞凋亡率均低于对照组,P<0.05。与BMSCs组和上清组比较,抗体组的凋亡率显著增高,P<0.05。抗体组12 h的凋亡率与24 h的凋亡率比较,差异有统计学意义,P<0.01。与BMSCs组和上清组比较,抗体组survivin、bcl-2基因的表达均有降低,P<0.05。结论：BMSCs对白血病细胞有保护作用,VLA-4抗体能够抑制白血病BMSCs与白血病细胞间的粘附,促进白血病细胞的凋亡。［中国当代儿科杂志,2010,12（11）：897-901］