CD34+ cells and CD34+CD38- subset from mobilized blood show different patterns of adhesion molecules compared to those from steady-state blood, bone marrow, and cord blood

As suggested previously, a down-regulation of some cellular adhesion molecules (CAMs) on CD34(+) hematopoietic progenitor cells (HPC) may contribute to their egress from bone marrow (BM) to peripheral blood (PB) by decreasing their adhesion to BM stromal cells. Besides counting the percentage of CAM-positive cells, we decided to define clearly the antigen density (AgD) of the CAM on mobilized- and steady-state CD34(+) HPC using QIFIKIT calibration beads. Five sources of cells were compared: PB and BM from normal donors (nPB, nBM) cord blood (CB), mobilized PB obtained from leukapheresis products (LKP), and mobilized BM (mBM) samples. In our study the CAM-AgD was the lowest on CD34(+) cells in LKP which, on the contrary, contained the highest percentage of CD117(+), CD54(+), CD58(+) cell subsets. As for CB, a greater proportion of CD44(+) and CD62L(+) cells was observed in LKP than in other products. The LKP-CD34(+) cell population contained a greater percentage of CD11a(+) cells when compared to mBM, but the lowest percentage of CD49d(+) and CD49e(+) cells when compared to all products. The proportion of the CD34(+)CD38(-) immature subset expressing CD11a, CD44, CD54, or CD62L was greater in LKP than in mBM; the CD62L-AgD was higher in LKP than in mBM. This quantitative analysis clearly showed a downregulation of all CAM on LKP-CD34(+). The CD44, CD62L, CD11a, and CD54 AgD decrease appears to be specifically involved in the egress of the CD34(+) subsets into PB. The control of antigen density of these adhesion molecules is likely to be clinically important for effective mobilization of HPC as well as for rapid engraftment following HPC transplant.     

Quantification of nucleated red blood cells in allogeneic marrow graft and impact of processing on recovery

BACKGROUND: In allogeneic bone marrow transplantation (BMT), a higher nucleated and CD34+ cell dose has been reported to improve various outcomes. Other cell types, such as lymphocyte subsets, also influenced BMT results. While nucleated red blood cells (NRBCs) represent a subset of bone marrow (BM) cell subpopulation, the question of their quantification in BM grafts and the impact of BM processing on their recovery has not been addressed. STUDY DESIGN AND METHODS: In a prospective study on 77 BM products, NRBCs were enumerated by flow cytometry and the recovery analyzed after manipulation. Because NRBCs could compromise white blood cell count, the impact of NRBC count on CD34+ cell percentage and total nucleated cell (TNC) dose were also determined. RESULTS: The mean percentage of NRBCs in BM grafts was 21.6 percent (range, 7.8%-40.9%). Mean NRBC recoveries after BM concentration or RBC depletion were 98.4 and 28.7 percent, respectively, close to those obtained for TNC cells (88.6 and 31.3%, respectively). When corrected with NRBC count, the mean percentages of corrected CD34+ cell and TNC dose were significantly modified when compared with uncorrected values, whatever the type of BM manipulation. CONCLUSION: Our data show that NRBC quantification might be of importance to improve quality control of BM products and to evaluate the influence of NRBCs cell dose on outcomes after BMT.     

p27基因下调对人骨髓和脐血造血祖细胞体外活性的影响

目的比较干扰p27基因表达对骨髓和脐血来源造血祖细胞增殖和造血潜能的影响,并探讨其相关机制。方法以含p27全长反义cDNA逆转录病毒感染经流式细胞仪分选的人骨髓与磁珠分离获得的人脐血来源的CD34+细胞,在含混合生长因子条件下体外培养不同时间,分别观察细胞生长曲线,检测细胞周期,确定细胞增殖能力;半固体培养计数集落形成,确定其造血能力;Westernblot检测p27与CDK2蛋白表达,明确基因转染效果并探讨p27反义cDNA促进细胞扩增的作用途径。以含p27全长正义cDNA和仅含绿色荧光蛋白(GFP)基因的病毒感染细胞为对照。结果与GFP和p27正义cDNA比较,p27反义cDNA对脐血造血祖细胞生长有明显促进作用(P<0.01),至培养第9天p27反义cDNA、p27正义cDNA和GFP组细胞数分别增加了(197.3±47.7)倍、(12.7±8.1)倍和(41.8±30.6)倍;骨髓造血祖细胞数增加不明显,至培养第9天3组细胞数分别增加了(36.0±22.3)倍、(8.7±6.8)倍和(14.1±10.4)倍。p27反义cDNA主要促进脐血和骨髓造血祖细胞S期增加,p27反义cDNA、p27正义cDNA和GFP组脐血造血祖细胞S期细胞百分比为(17.0±4.8)%,(2.0±0.8)%和(4.1±1.8)%;骨髓造血祖细胞则为(8.4±4.4)%,(1.0±0.7)%和(3.8±1.4)%。p27反义cDNA对骨髓和脐血的集落形成能力促进作用高于GFP组(P<0.0     

G-CSF对慢性粒细胞白血病患者bcr/abl+-CD34+细胞增殖、分化的影响

目的了解G-CSF对bcr／abl^＋-CD34^＋细胞增殖、分化的影响。方法采集慢性粒细胞白血病（CML）患者的bcr／abl^＋-CD34^＋细胞，分别以0、10、100、1000ng／ml的G-CSF与之共培养，并以正常骨髓CD34’细胞为对照，通过锥虫蓝拒染法、流式细胞术和光学显微镜观察研究其细胞增殖、周期分布、抗原分化和形态变化特点。结果所有实验组bcr／abl^＋-CD34^＋细胞均明显增长，其中G-CSF10ng／ml组培养48、96h，其细胞数显著高于同期无G-CSF组（P〈0．05）；而正常CD34^＋细胞数只在G-CSF存在的情况下增长明显，其中G-CSF100ng／ml组培养48、96、144h细胞数均显著高于同期无G-CSF组（P值分别为〈0．05，0．01，0．01）；G-CSF10、100、1000ng／ml组的bcr／abl^＋-CD34^＋细胞培养144h其Go／G1期比例显著低于G-CSF空白组（P〈0．05）；而正常CD34^＋细胞G-CSF10、100、1000ng／ml组培养48和96h其Go／G1期比例均明显低于无G-CSF组（P〈0．01）；bcr／abl^＋-CD34^＋细胞及正常CD34^＋细胞CD34抗原表达均随培养时间延长而下降，伴随CD33和CDl3抗原先升后降，其变化与G-CSF浓度无关。但bcr／abl^＋-CD34^＋细胞的各抗原分化显著快于正常CD34^＋细胞。bcr／abl^＋-CD34^＋细胞和正常CD34^＋细胞均随着增殖分化表现出终末细胞的形态特征。结论G-CSF能促进bcr／abl^＋-CD34^＋细胞及正常CD34^＋细胞的增殖，但并非前者增殖的必要条件。bcr／abl^＋-CD34^＋细胞比正常CD34^＋细胞分化更快，但两类细胞的分化速度均与G-CSF浓度无关。     

骨髓增生异常综合征患者骨髓CD34-和CD34+细胞凋亡与增殖的意义及对生存的影响

本研究分析骨髓增生异常综合征（MDS）患者骨髓CD34^＋和CD34-细胞凋亡和增殖情况,探讨MDS的发病机制,并判断其与疾病预后的关系。流式细胞术分析20例高危MDS、20例低危MDS患者及10例正常对照者骨髓CD34^＋细胞的比例、CD34^＋细胞和CD34-细胞凋亡、增殖的百分率,计算各组中的凋亡／增殖（A／P）比。并用单变量和多变量生存分析法分析CD34^＋细胞和CD34-细胞增殖和凋亡对预后的影响。结果表明：①MDS高危组患者骨髓CD34^＋细胞的比例明显高于低危组（P〈0．05）,而低危组与对照组比较无显著差异;②CD34^＋,CD34-细胞的凋亡率在MDS低危组中均为最高,明显高于MDS高危组和对照组。在低危组中,CD34-细胞的凋亡率为（80．36±1．82）％,明显高于CD34^＋细胞的（54．75±2．18）％（P〈0．05）,而在高危组中,CD34^＋,CD34-细胞的凋亡率无显著差异;③CD34^＋细胞的增殖率在MDS高危组中最高,明显高于低危组和对照组,而CD34-细胞的增殖率在MDS高危和低危组间无显著差异。高危组CD34^＋细胞的增殖率为（50．67±3．37）％,明显高于CD34-细胞的（30．99±1．96）％（P〈0．05）;④CD34^＋、CD34-细胞的A／P值在MDS低危组均明显高于高危组和正常对照组,而CD34-细胞的A／P值在MDS高、低危组均明显高于CD34^＋细胞的A／P值（P〈0．05）。此外,CD34^＋细胞的凋亡率与生存及预后明显相关。结论：CD34^＋细胞百分率随MDS危险度增加而逐渐增加,在低危组中以CD34-细胞的凋亡占主导,随着病情进展,在高危组中则以CD34^＋细胞的增殖占主导,提示异常的凋亡和增殖在MDS的发生和发展中起重要作用。此外,CD34^＋细胞的凋亡率可能是独立的预后不良因素,抑制凋亡可能诱导MDS向白血病的转化。     

Use of merocyanine 540 for the isolation of quiescent, primitive human bone marrow hematopoietic progenitor cells.

Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle. We rationalized that MC540 uptake could be used to distinguish between quiescent and metabolically active cells and, therefore, to fractionate normal and leukemic BM cells and normal mobilized peripheral blood (MPB) cells into functionally distinct groups of progenitors. BM and MPB cells were separated into fractions ranging in fluorescence from MC540Bright to MC540Dim. Cell cycle analysis of these fractions revealed that the MC540Dim fraction of normal and CML BM CD34+ cells constituted the most quiescent fraction, and the MC540Bright fractions from these cell types contained the most actively cycling cells. However, no differences in the percentage of cells in G/G1 were observed between MC540Bright and MC540Dim fractions of MPB CD34+ cells. To investigate if these cell cycle status differences translated into distinct functional properties, the hematopoietic potential of BM CD34+MC540Bright and CD34+MC540Dim cell fractions was analyzed in vitro in long-term BM cultures and limiting dilution analysis (LDA) assays. CD34+MC540Dim cells produced more total and committed progenitor cells in long-term cultures than did the CD34+MC540Bright fraction. The CD34+MC540Dim fraction also contained a 2-fold higher number of long-term hematopoietic culture-initiating cells (LTHCIC) than the CD34+MC540Bright fraction, as defined by LDA assays. These data demonstrate that MC540 can be a useful probe for the isolation of primitive HPC from some hematopoietic tissues and may assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation of HPC.     

Predictive value of the original content of CD34(+) cells for enrichment of hematopoietic progenitor cells from bone marrow harvests by the apheresis procedure

We retrospectively investigated the feasibility of the apheresis procedure for red blood cell (RBC) reduction with a closed-bag system. We also sought to determine the optimal processing volume for the maximal recovery of hematopoietic progenitor cells (HPC). Twelve bone marrow (BM) harvests were processed for major ABO-incompatible allogeneic transplantation and one BM harvest was processed for autologous transplantation. The processing was performed through seven apheresis cycles with a two-bag system using COBE Spectra Version 6.1. The mean recovery rates were compared in the products after four cycles and seven cycles of BM processing. Mean cell recovery rates were 79.2% (67.6-97.5%) and 87.3% (68.9-111.9%) for the mononuclear cells (MNC) and 84.5% (69.4-109.5%) and 92.0% (79.0-107.7%) for the CD34(+) cells after four and seven cycles, respectively. A mean of 96.3% (93.0-98.1%) of the RBCs were finally removed. The yield of CD34(+) cells after seven cycles of processing (median: 10.35 x 10(7) cells) was 7.9% greater than that after four cycles of processing (median: 9.65 x 10(7) cells), exhibiting a less-than-significant enhancement in yield. The CD34(+) cell contents recovered in the concentrates up to four cycles (r = 0.989) and up to seven cycles (r = 0.993) were strongly correlated with the original content of the CD34(+) cells. Engraftment was obtained in all patients except one patient infused with purified CD34(+) cells. This latter result confirmed the hematopoietic potential of the cell populations recovered. Granulocyte recovery (defined as an absolute neutrophil cell count > or = 500/microL for a period of three consecutive days) ranged from 8 to 25 days (median: 16 days) post-transplantation. No hemolytic reaction was observed in any of the patients. Our results confirmed the efficacy of BM processing cycles with the COBE Spectra device. However, we could not conclude that the large-volume apheresis for BM processing significantly enhanced the yields of HPC. The final recovery of CD34(+) cells after processing could be predicted from the CD34(+) cell content of the original collected marrow.     

人急性B淋巴细胞白血病-NOD/SCID/IL2rγnull新生小鼠异种移植模型的建立

本研究旨在应用NOD/SCID/IL2rγnull新生小鼠建立人急性B淋巴细胞白血病(B-ALL)的异种移植模型。NOD/SCID/IL2rγnull新生期(出生48 h之内)小鼠经亚致死剂量(100 cGy)137Cs全身照射后,由面静脉输注FACSAria流式细胞仪分选纯化的B-ALL患者骨髓CD3-CD4-CD8-CD34+CD19+细胞,于移植后8-12周用流式细胞术检测受鼠外周血、骨髓和脾脏中人源细胞的植入水平及其免疫表型,并通过HE染色评价人源B-ALL细胞在受鼠各组织器官中的迁移浸润能力。结果表明,在接受B-ALL患者CD34+CD19+细胞移植的受鼠外周血、骨髓和脾脏细胞中,不仅检测到了不同程度的人源B-ALL(huCD45+CD19+)细胞的植入〔(83.36±10.05)%,(93.88±5.05)%,(88.31±5.01)%〕,而且植入细胞具有与B-ALL患者相似的细胞形态和免疫表型特征。此外,人源B-ALL细胞广泛迁移浸润到受鼠的肝脏、肺脏、肾脏和脑等组织器官中。结论:NOD/SCID/IL2rγnull新生小鼠模型能够支持B-ALL患者CD34+CD19+细胞的高水平植入,是对人B-ALL进行体内功能性研究的新型异种移植模型。     

Prospective flow cytometric evaluation of nucleated red blood cells in cord blood units and relationship with nucleated and CD34(+) cell quantification

BACKGROUND: Cord blood (CB) represents an alternate source of stem cells in transplantation. Nucleated red blood cells (NRBCs) are a physiological subset of CB population. Although it is important to have an accurate estimate of CD34(+) cell number, NRBCs could compromise white blood cell count and interfere with CD34(+) cell quantification. STUDY DESIGN AND METHODS: A total of 826 CB units were analyzed for total nucleated cells (TNCs), NRBCs, and CD34(+) cells by flow cytometry. NRBCs were also counted conventionally by manual microscopy. Percentages of CD34(+) cells corrected by NRBC count (CD34+c) were determined as follows: %CD34+c = CD34(+)/CD45(+) (x10(6))/(TNCs (x10(8)) - NRBCs (x10(8))). RESULTS: The mean percentages of CD34+ cells and NRBCs were 0.27 percent (range, 0.01%-1.25%) and 7.64 percent (range, 0.13%-84%), respectively. Comparison between flow cytometric and microscopic NRBC count showed a regression of y = 0.685 + 0.719x and a coefficient of determination of r(2) = 0.721. When corrected with NRBC count, the mean percentage of CD34(+) c cells was 0.295 percent (p = 0.0008 compared with CD34(+)%) and mean TNCc count was 14.8 x 10(8) (p < 10(-4) compared to TNC count). CONCLUSION: The determination of NRBCs with a flow cytometric method might represent a new strategy for providing satisfactory quality assurance controls of CB products.     

Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow: reference patterns for age-related changes and disease-induced shifts

BACKGROUND: The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainings in flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal hematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormal differentiation lineage. METHODS: For a quick and easy flow cytometric screening of BM samples, we selected six quadruple immunostainings that cover multiple differentiation stages of the B-cell, monocytic, granulocytic, and erythroid lineages: TdT/CD20/CD19/CD10 and CD45/CD34/CD19/CD22 for B cells, CD34/CD117/CD45/CD13.33 for precursor granulocytic and precursor monocytic cells (myelo/monoblasts), CD14/CD33/CD45/CD34 for monocytic cells, CD16/CD13/CD45/CD11b for granulocytic cells, and CD71/CD235a/CD45/CD117 for erythroid cells. RESULTS: The six quadruple immunostainings reveal specific staining patterns in normal BM, which allow the recognition of various subpopulations of the respective lineages. These staining patterns can be used as a frame of reference for recognition of normal and abnormal BM development. Examples of normal (age-related) variations in these otherwise stable staining patterns are presented together with several abnormal differentiation patterns. CONCLUSIONS: Although alternative immunostainings can be used (e.g., including NK- and T-cell markers), we feel that the selected six stainings represent a comprehensive and easy screening phase for quick identification of shifts in the composition of the studied differentiation lineages, reflecting age-related changes or disease-induced BM abnormalities.     

阵发性睡眠性血红蛋白尿症患者外周血淋巴细胞增殖、杀瘤活性及其对正常表型骨髓造血细胞的影响

阵发性睡眠性血红蛋白尿症 (PNH)克隆可能由于缺乏GPI锚连蛋白而逃脱免疫攻击 ,形成生长优势。本研究测定PNH患者淋巴细胞增殖反应及对K5 6 2细胞的杀伤作用 ,并与正常对照比较 ,观察其淋巴细胞功能。采用体外液体培养体系 ,应用免疫磁珠技术分选PNH患者骨髓CD34+ 及CD34- 细胞 ,分别向 2组细胞及对照细胞加入自身CD5 9+ 或CD5 9- 淋巴细胞及其培养上清液、外源性IFN γ及IL 2。培养 10天后 ,测定骨髓细胞中PNH细胞 (CD5 9- )百分数 ,观察淋巴细胞对骨髓中CD34+ 及CD34- 细胞中PNH细胞含量的影响。研究结果表明 ,对PHA的增殖反应 ,PNH患者未分选的淋巴细胞与健康对照比较、PNH患者自身CD5 9+ 与CD5 9- 淋巴细胞之间比较均无统计学差异 ,但对K5 6 2细胞的杀伤作用PNH患者未分选的淋巴细胞明显低于健康对照组 ,分别为 (5 0 .0 0± 2 8.6 7) %及 (76 .13± 10 .15 ) % (P <0 .0 5 ) ,而PNH患者CD5 9- 淋巴细胞与CD5 9+ 淋巴细胞之间无显著性差异。PNH患者骨髓细胞中加入自身CD5 9- 淋巴细胞或其培养上清液、CD5 9+ 淋巴细胞或其培养上清液、外源性IFN γ和IL 2培养后 ,CD5 9+ 细胞均有不同程度下降 ,除CD5 9+ 淋巴细胞组外 ,其他各组CD5 9+ 细胞下降与培养前比较有显著性差异 (P <0 .0 5 ) ,IFN γ及IL 2组     

Increased Nucleated RBCs in Cord Blood: Not an Exclusion Criterion but a Quality Indicator for Hematopoietic Progenitor Cell Transplantation

Increased nucleated red blood cell (NRBC) counts have been reported to be associated with adverse fetal outcomes, and cord blood units (CBUs) with increased NRBC counts require a 2^nd questionnaire to determine their suitability for transplantation. However, a recent study demonstrated a positive correlation of NRBCs with CD34⁺ cells and total nucleated cells (TNCs). We evaluated the association between the NRBC count and hematopoietic progenitor cell (HPC) content (TNC and CD34⁺ cell counts) in Korean full-term newborn CBUs. In addition, we assessed whether an increased NRBC count is associated with newborn health problems that impair CBU safety. Among the 32,876 units processed from May 2006 to December 2018, a total of 23,385 CBUs with a TNC count ≥ 7 × 10⁸ and reliable perinatal information were analyzed to assess the association of the NRBC count with CBU parameters, and the newborns associated with 457 CBUs that required the 2^nd questionnaire due to an increased NRBC (≥ 15 NRBCs/100 WBCs) were assessed at one year for health problems that threatened CBU safety. The majority of the CBUs that required the 2^nd questionnaire due to an increased NRBC count (96.9%) were determined to be suitable for transplantation. Those with an increased NRBC count showed significantly higher CD34⁺ cell and TNC counts and a higher rate of transplantation (P < 0.001, < 0.001 and 0.025, respectively). NRBCs showed a significant positive correlation with TNCs and CD34⁺ cells and a significant negative correlation with birth weight (all P < 0.001; adjusted r = 0.185, 0.369 and - 0.029, respectively). In the multiple linear regression analysis, NRBCs showed independent and positive correlations with TNCs and CD34⁺ cells after adjustments for birth weight and gestational age (all P < 0.001; β = 0.182, adjusted R² = 0.053 and β = 0.367, adjusted R² = 0.418). An increased NRBC count in full-term normal delivery is a surrogate marker of HPCs in CBUs rather than an exclusion criterion for CBU safety. Moreover, providing the NRBC count together with the NRBC-corrected TNC count will be useful for clinicians to select CBUs for transplantation.     

Establishment of mouse embryonic fibroblast cell lines that promote ex vivo expansion of human cord blood CD34+ hematopoietic progenitors

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic progenitor cells (HPC) is vital to stem cell transplantation. The use of a monolayer of stromal cells on which to grow HPC in direct contact allows high efficiency ex vivo expansion of HPC. Here, we report an establishment of three murine embryonic fibroblast stromal cell lines from adherent cells of day-12 mouse embryos. Among them, HYMEQ-5 was most efficient in supporting long-term maintenance of human umbilical cord blood (CB) CD34(+) cells. Human CB CD34(+) cells cultured on HYMEQ-5 in the presence of stem cell factor (SCF), thrombopoietin, and flk-ligand (FL) showed high expansion of CD34(+)CD38(-) cells and highly proliferative potential-colony forming cells (HPP-CFC). Direct cell-to-cell contact between CD34(+) cells and HYMEQ-5 was important for this expansion. RT-PCR analysis showed that HYMEQ-5 produced FL, SCF, interleukin-6, and macrophage colony-stimulating factor (M-CSF). Expanded CB CD34(+) cells efficiently reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings suggest that HYMEQ-5 provides a milieu that supports long-term human hematopoiesis as well as ex vivo expansion of human CB CD34(+) HPC. This cell line may facilitate elucidation of the mechanism of cellular interactions between HPC and stromal cells.     

Tumor necrosis factor alpha cooperates with interleukin 3 in the recruitment of a primitive subset of human CD34+ progenitors

We have recently demonstrated that tumor necrosis factor alpha (TNF- alpha) potentiates interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor-induced growth of CD34+ hematopoietic progenitor cells (HPC), and favors the generation of dendritic/Langerhans cells. The stimulatory effect of TNF-alpha was detailed in the present study. Thus, CD34+ HPC entering in cycle (S/G2M) after a 48-h pulse with IL-3 expressed the transferrin receptor (TfR), and fluorescence-activated cell sorter-separated TfR+ HPC, but not TfR-HPC, showed a high proliferative response to IL-3. In contrast, TfR-HPC were found to undergo strong proliferation in response to IL-3 + TNF-alpha. Limiting dilution experiments indicated that TNF-alpha increased both the frequency and the average size of clones generated from TfR-HPC as a result of the development of a higher number of large clones. In contrast, TNF-alpha did not enhance the IL-3-dependent proliferation of TfR+ HPC. Preculturing CD34+ HPC for 48 h with TNF- alpha enhanced the subsequent generation of IL-3-dependent colony- forming units. Precultures with TNF-alpha or cultures with suboptimal doses of TNF-alpha allowed the recruitment of cells with both granulocytic and monocytic differentiation potential. Taken together, our results indicate that TNF-alpha recruits a subpopulation of CD34+ HPC hyposensitive to IL-3, with high proliferative capacity and some features of multipotential progenitors, that are likely to be more primitive than those responding to IL-3 alone.     

地中海贫血患儿血清群体反应性抗体对脐血CD34~+细胞增殖和凋亡的影响

本研究探讨特异性群体反应性抗体(panel reactive antibody,PRA)对脐血CD34+细胞的影响。取含PRA的β地中海贫血患儿血清,与脐血CD34+细胞、补体联合孵育,观察其对CD34+细胞的影响,并以[3H]TdR掺入法测定细胞DNA合成及流式细胞仪检测细胞凋亡。结果表明,PRA血清组细胞培养上清中乳酸脱氢酶水平高于对照组;PRA血清组脐血CD34+细胞DNA合成能力较对照组下降。流式细胞仪检测显示,各实验组间脐血CD34+细胞凋亡率差异无统计学意义。结论:特异性PRA血清对脐血CD34+细胞的增殖有抑制作用,补体可增强上述作用;特异性PRA血清对脐血CD34+细胞的凋亡无明显影响。     

间充质干细胞对多发性骨髓瘤细胞生长及硼替佐米诱导其凋亡的影响研究

目的 探讨间充质干细胞在多发性骨髓瘤细胞生长以及硼替佐米诱导骨髓瘤细胞凋亡中的作用.方法 取15例临床确诊的多发性骨髓瘤(MM)患者以及3名正常供者的骨髓标本,分离其间充质干细胞(MM-BMSC和ND-BMSC);测定细胞生长曲线、免疫表型和细胞因子分泌水平.将骨髓瘤细胞(NCI-H929)与BMSC细胞共培养,并加入蛋白酶体抑制剂硼替佐米,观察BMSC对NCI-H929细胞生长增殖的影响;进一步通过Annexin V-FITC/PI双染法流式细胞术(FCM)检测BMSC对硼替佐米诱导NCI-H929细胞凋亡的影响.结果 成功分离得到MM患者以及正常供者骨髓间充质干细胞,FCM检测显示两者均高表达CD73和CD105(＞95%),表达CD44和CD29,不表达CD31、CD34、CD45和HLA-DR(＜1%)等表面分子.生长曲线测定显示MM-BMSC增殖较为缓慢,倍增时间(82 h)较ND-BMSC(62 h)略有延长(P＜0.05).与ND-BMSC比较,MM-BMSC分泌高水平的细胞因子IL-6和VEGF,分别为(188.8±9.4)pg/ml对(115.0±15.1)pg/ml和(1497.2±39.7)pg/ml对(1329.0±21.1)pg/ml.将BMSC与骨髓瘤细胞NCI-H929共培养,MM-BMSC可促进骨髓瘤细胞的生存,降低NCI-H929细胞对硼替佐米的敏感性,明显抑制硼替佐米诱导的瘤细胞凋亡.结论 MM-BMSC可促进骨髓瘤细胞的生长,明显减少蛋白酶体抑制剂硼替佐米诱导的细胞凋亡.     

CD44单克隆抗体诱导THP-1细胞分化和凋亡作用及其机制探讨

目的 观察CD44单克隆抗体A3D8对急性单核细胞白血病细胞系THP-1细胞的增殖抑制作用,并探讨其作用途径.方法 采用MTT法检测A3D8对THP-1细胞的增殖抑制效应;流式细胞术分析THP-1细胞CD33、CD15、CD11b、CD14、Annexin-V、caspase-3、细胞周期;Western blot法分析p-Akt、磷酸化细胞外调节蛋白激酶(P-ERK)、bcl-2和p27kip1蛋白的表达.结果 A3D8能显著抑制THP-1 细胞增殖,呈剂量和时间的量效关系.2.0 μg/ml A3D8作用THP-1细胞1～6 d后细胞明显分化.A3D8处理4 d,THP-1细胞CD33、CD15、CD11b、CD14表达水平与对照组比较[平均荧光强度(MFI)]分别为68.9±2.0对39.3±1.5、61.7±5.5对12.9±2.6、67.3±3.8对14.0±2.0、83.0±5.7对8.0±1.0(P均<0.01).细胞周期阻滞在G0/G1期.处理4 d实验组和对照组p-Akt、p-ERK、bcl-2蛋白分别为0.24±0.06对1.20±0.15、0.32±0,05对1.24±0.09、0.11±0.05对0.65 ±0.07,实验组较对照组显著减少(P均<0.01);处理4 d实验组和对照组p27kipl.蛋白分别为1.08±0.09对0.10±0.02,实验组较对照组显著增加(P<0.05).A3D8处理5 d THP-1细胞Annexin-V、caspase-3阳性率与对照组比较,分别为(32.5±2.5)%对(2.4±0.3)%、(33.3±2.5)%对(3.6±0.3)%(P均<0.01).结论 CD44单克隆抗体A3D8可能通过抑制PI3K/Akt和ERK1/2信号通路诱导THP-1细胞分化和凋亡.

Abstract：

Objective To investigate the effects of anti-CD44 mAb A3D8 on the cell proliferation of human acute monocytic leukemia cell line THP-1 and its mechanism. Methods Cell proliferation was assayed with MTT method, the expression of CD33, CD15, CD11b, CD14, Annexin-V, caspase-3 and cell cycle with flow cytometry, and the expression of p-Akt, p-ERK, bcl-2 and p27kipl with Western blot. Results A3D8 could remarkably inhibit the proliferation capacity of the THP-1 cells in a dosage- and time-dependent manner. THP-1 differentiation was observed when treated with A3D8 (2.0 μg/ml) for one to six days. Expression of CD33 (68. 9 ±2.0 vs 39.3 ± 1.5), CD15(61. 7 ±5.5 vs 12.9 ±2.6), CD11b (67.3 ± 3.8vs 14.0±2.0) and CD14 (83.0 ±5.7 vs 8.0 ± 1.0) was significantly increased at day 4 compared with the control group ( all P < 0.01). Cell cycle of the THP-1 cells was arrested in G1/G1. Expression of the Annexin-V [(32.5±2.5)% vs (2.4±0.3)%] and caspase-3 [(33.3 ±2.5)% vs (3.6±0.3)%] was much higher than that in normal controls (all P <0. 01) , and apoptosis was observed in THP-1 cells at day 5. Expression of p-Akt (0.24 ±0.06 vs 1.20 ±0.15), p-ERK (0. 32 ±0.05 vs 1. 24 ±0. 09), and bcl-2 (0. 11 ±0.05 vs 0. 65 ± 0. 07) was much lower than that of the controls ( all P < 0. 01), while p27kipl (1.08 ± 0.09 vs 0. 10 ± 0.02) was significantly increased at day 4 (P < 0.05). Conclusion Anti-CD44 antibody can induce the differentiation and apoptosis of THP-1 cell through inhibiting PI3K/Akt and ERK1/2 signaling pathway.     

脐血造血干/祖细胞免疫表型的流式细胞术双标法分析及其意义

目的比较脐血和骨髓中造血干/祖细胞(HSPC)的免疫表型差异.方法使用流式细胞术(FCM)双标法对38份脐血及10份骨髓HSPC进行免疫表型分析.结果①脐血有核细胞中CD34+细胞所占比例与骨髓中相近,约为0.5%;②脐血CD34+细胞中CD34+CD38-[(17.C4±5.37)%]、CD34+HLA-DR-[(32.65±10.71)%]及CD34+H-CAM+(CD44+)[(77.84±7.69)%]亚群含量均高于骨髓[含量分别为(8.26±3.19)%、(14.05±1.67)%和(70.02±6.40)%],CD34+CD13+、CD34+CD19+亚群比例低于骨髓.结论脐血与骨髓CD34+细胞比例相近,但前者较原始的干细胞含量更高,故脐血是极具潜力的HSPC来源;而脐血CD34+细胞中髓系及淋系祖细胞含量低于骨髓,可能是脐血移植后造血及免疫重建缓慢的原因之一.