TBTC induces adipocyte differentiation in human bone marrow long term culture

Organotins are widely used in agriculture and the chemical industry, causing persistent and widespread pollution. Organotins may affect the brain, liver and immune system and eventually human health. Recently, it has been shown that tri-butyltin (TBT) interacts with nuclear receptors PPAR gamma (peroxisome proliferator-activated receptor gamma) and RXR (retinoid x receptor) leading to adipocyte differentiation in the 3T3 cell line. Since adipocytes are known to influence haematopoiesis, for instance through the expression of cytokines and adhesion molecules, it was considered of interest to further study the adipocyte-stimulating effect of TBTC in human bone marrow cultures. Nile Red spectrofluorimetric analysis showed a significant increase of adipocytes in TBTC-treated cultures after 14 days of long term culture. Real-time PCR and Western blot analysis confirmed the high expression of the specific adipocyte differentiation marker aP2 (adipocyte-specific fatty acid binding protein). PPAR gamma, but not RXR, mRNA was increased after 24 h and 48 h exposure. TBTC also induced a decrease in a number of chemokines, interleukins, and growth factors. Also the expression of leptin, a hormone involved in haematopoiesis, was down regulated by TBTC treatment. It therefore appears that TBTC induced adipocyte differentiation, whilst reducing a number of haematopoietic factors. This study indicates that TBTC may interfere in the haematopoietic process through an alteration of the stromal layer and cytokine homeostasis.     

Mercury toxicity to hemopoietic and tumor colony-forming cells and its reversal by selenium in vitro.

Inorganic and organic mercury, in micromolar concentrations, inhibited colony formation in primary cultures of mouse bone marrow and in P815 mouse mastocytoma cultures. When selenium, in the form of selenous acid, was added to cell cultures, it was able to prevent the inhibition of colony formation caused by continuous exposure to inorganic mercury in P815 cell cultures and primary cultures of bone marrow. Selenite was also able to prevent colony inhibition resulting from continuous exposure to methylmercury in P815 cell cultures, but failed to prevent colony inhibition in primary cultures of bone marrow. In consideration of the overall effect of methylmercury, the bone marrow is a potential site of toxicity.     

7,12-Dimethylbenz[a]anthracene-induced bone marrow toxicity is p53-dependent.

Polycyclic aromatic hydrocarbons (PAHs) are known immunotoxins and carcinogens. Our laboratory and others have demonstrated that metabolism of these compounds by CYP1B1 is required for carcinogenicity and immunotoxicity to occur. Previously, our laboratory reported significantly decreased bone marrow cellularity in mice following 7,12-dimethlybenz[a]anthracene (DMBA) administration. In addition, we have observed that DMBA causes apoptosis via activation of both caspase-8 and -9 in pre-B cells co-cultured with bone marrow stromal cells in vitro. In this study, we investigated the importance of the p53 protein in the bone marrow response to DMBA. Through the use of p53 gene knockout mice, we demonstrated that the effect of DMBA on bone marrow cellularity is p53-dependent. In addition, apoptosis of primary cultures of progenitor B cells cultured with bone marrow stromal cells and DMBA is also p53-dependent. The results of this study provide evidence for the importance of p53 in the signaling pathways by which PAHs cause immunotoxicity.     

Triorganotin-induced cytotoxicity to rat thymus, bone marrow and red blood cells as determined by several in vitro assays

To further investigate the immunotoxic effects of tri-n-propyltin chloride (TPTC), tri-n-butyltin chloride (TBTC) and triphenyltin chloride (TPhTC) several cytotoxicity tests with a series of trialkyltin chlorides and TPhTC were carried out, using isolated rat thymocytes as target cells. Thymocytes, cultured in a serum-supplemented medium, were exposed to organotin concentrations ranging from 0.01 to 10 microM for periods up to 30 h. Parameters such as cell count, trypan blue exclusion, chromium release, thymidine incorporation and cyclic AMP production were used to evaluate the cytotoxicity of these compounds. The more lipophilic compounds TPTC, TBTC, tri-n-hexyltin chloride (THTC) and TPhTC appeared most cytotoxic, reducing thymidine incorporation at concentrations as low as 0.05-1 microM. Membrane damage as determined by trypan blue exclusion and chromium release occurred at higher levels (1-10 microM). The water soluble homologue trimethyltin chloride (TMTC) was least effective in all test models. When phosphate-buffered saline supplemented with glucose was used as incubation medium, TBTC appeared more cytotoxic to thymocytes. Using this medium in 5-h incubations the cytotoxicity of TBTC to thymus, bone marrow and red blood cells was compared. Bone marrow cells were slightly less sensitive than thymocytes, while red cells were relatively resistant. In conclusion, of the triorganotin compounds especially the lipophilic homologues are cytotoxic in vitro.     

Metabolism of doxorubicin in long-term bone marrow cultures and SR-4987 stromal established cell line

The metabolism of doxorubicin was studied in murine long-term bone marrow cultures (LTBMC) and in SR-4987 established stromal cells in comparison with primary cultures of murine and rat hepatocytes. The toxicity of metabolites was verified by testing their effects on the clonogenicity of granulo-macrophage progenitors. Metabolic activity was compared in subcellular fractions of SR-4987 cells and murine hepatocytes. Doxorubicin was transformed in long-term bone marrow cultures, SR-4987 cells and murine/rat hepatocytes to less toxic metabolites: 13-OH doxorubicin and a less polar metabolite which were non-toxic on granulo-macrophage progenitors. Among the hemopoietic compartments, stromal cells were responsible for the biotransformation of doxorubicin. The capability of the SR-4987 established stromal cell line to metabolize doxorubicin was higher than that of primary cultures of hepatocytes and bone marrow, and the highest activity was concentrated in the microsomes. These results suggest that in vitro models using primary cell cultures and established cell lines could be a useful tool for investigating the mechanisms underlying detoxification in the bone marrow stromal population.     

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1alpha,25-dihydroxyvitamin D(3) and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 microM) completely inhibited the calcium release induced by IL-1beta, TNF-alpha, and LPS. The resorptive effect of 1alpha,25-dihydroxyvitamin D(3) was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E(2). Celecoxib (0.1 microM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE(2) in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1beta, TNF-alpha, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.     

全反式维甲酸对人骨髓基质细胞分泌GM-CSF的影响

目的:探讨全反式维甲酸对骨髓基质细胞分泌GM-CSF的影响.方法:原代培养正常人和急性早幼粒细胞白血病患者的骨髓基质细胞,ELISA法检测不同浓度维甲酸对正常和急性早幼粒细胞白血病骨髓基质细胞分泌GM-CSF的差异.结果:不同浓度全反式维甲酸对正常和急性早幼粒细胞白血病骨髓基质细胞分泌GM-CSF浓度存在差异,维甲酸呈浓度依赖性调高正常和急性早幼粒细胞白血病患者骨髓基质细胞分泌GM-CSF浓度.结论:维甲酸可能通过骨髓基质细胞分泌GM-CSF而促进造血,间接诱导细胞分化治疗.     

In vitro hematotoxicity of Aplidine on human bone marrow and cord blood progenitor cells.

Aplidine is a cyclic depsipeptide that was isolated from a Mediterranean marine tunicate, Aplidium albicans. In experimental animals, Aplidine mediated an in vivo inhibitory effect in a number of tumor cell types. In humans, Aplidine is currently used in phase I clinical trials. Aiming to predict the hematotoxicity of Aplidine in humans, samples from human bone marrow (BM) and cord blood (CB) were exposed in vitro to increasing concentrations of the drug and then assayed for the clonogenic ability of myeloid (CFU-GM), erythroid (BFU-E), megakaryocitic (CFU-Meg) and pluripotent (CFU-Mix) hematopoietic progenitors. We investigated whether predictions of the hematotoxicity of Aplidine based on bone marrow (BM) cultures were reproduced when a more readily available source of human hematopoietic cells, cord blood cells, was used in experiments involving 24-h exposures. Although hematopoietic progenitors derived from bone marrow were generally more sensitive than those derived from cord blood, differences on the IC50, IC70 and IC90 varied within a relatively small range of 1.6-6.2-fold. Moreover, data obtained from cord blood cultures confirmed the observation made in bone marrow assays indicating that the myeloid (CFU-GM) and the erythroid (BFU-E) progenitors were the least sensitive to Aplidine. Regardless of the origin of the hematopoietic progenitors (bone marrow or cord blood) the toxicity of Aplidine in human hematopoietic progenitors (IC50: 150-2250 nM) was lower than that observed in previous studies with tumoral cell lines.     

Role of mitogen-activated protein kinase family in serum-induced leukaemia inhibitory factor and interleukin-6 secretion by bone marrow stromal cells

1: In the haematopoietic microenvironment, bone marrow stromal cells play an important role in regulating haematopoiesis by expressing various cytokines, including leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6). However, the intracellular signal that regulates cytokine secretion in bone marrow stromal cells has not been determined. The aim of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) family in serum-induced secretion of LIF and IL-6 by bone marrow stromal cells. 2: Transformed human bone marrow stromal cells (HS-5) were stimulated with foetal calf serum (FCS) to produce LIF and IL-6. FCS also induced activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). 3: Both PD98059 (MAPK/ERK kinase inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated FCS-induced LIF protein production and gene expression. SB203580 decreased IL-6 production and gene expression, but PD98059 had no effect on IL-6 production and gene expression. 4: Expression of a dominant-negative mutant form of JNK1 that blocked FCS-induced JNK activity had no effect on protein production and gene expression of these cytokines. 5: These findings demonstrate that both ERK and p38 MAPK are involved in FCS-induced LIF secretion, whereas only p38 MAPK is important for IL-6 secretion, and that FCS-induced activation of JNK has no effect on the production of LIF and IL-6. We conclude that, in spite of their similar biological effects, they are differentially regulated at the level of MAPK activity in bone marrow stromal cells.     

7-12 Dimethylbenz[a]anthracene-induced bone marrow hypocellularity is dependent on signaling through both the TNFR and PKR

In addition to being carcinogenic, polycyclic aromatic hydrocarbons (PAHs) are known to cause deleterious effects on the immune system, including a marked reduction in bone marrow granulocytes and B lymphocytes. The molecular mechanisms underlying bone marrow hypocellularity are incompletely understood. Hematopoiesis is governed by the production of cytokines and the resultant signaling pathways that they initiate. Our hypothesis was that PAHs may disrupt cytokine production in the bone marrow resulting in the perturbation in bone marrow cellularity observed after PAH administration. TNF-alpha and IFN-gamma are two cytokines that are involved in the regulation of hematopoiesis. Based on observations made in previous research, we sought to determine if the effects of 7-12 dimethylbenz[a]anthracene (DMBA) on the murine bone marrow were mediated through the actions of these molecules. Transgenic mice that were null for either IFN-gamma or TNF-alpha receptors were injected with DMBA and the resulting bone marrow cellularity compared with wild-type mice. We observed that tumor necrosis factor alpha receptor (TNFR) null mice were protected against DMBA-induced bone marrow hypocellularity, while IFN-gamma null mice were not. In addition, we found that dsRNA-dependent protein kinase (PKR) null mice were also protected from DMBA-induced hypocellularity. PKR is an intracellular signaling molecule that has been demonstrated to be activated by TNFR-mediated signaling. Furthermore, we observed upregulation of PKR in the bone marrow after DMBA administration that was dependent on signaling through TNFR. These results point to a role for TNFR-dependent signaling, operating at least in part via PKR activation, as a mechanism for DMBA-induced bone marrow toxicity.     

Tributyltin chloride induces ABCA1 expression and apolipoprotein A-I-mediated cellular cholesterol efflux by activating LXRalpha/RXR

Organotins, including tri-butyltin chloride (TBTC), are widely used in agricultural and chemical industries and cause persistent and widespread pollution. TBTC has been shown to activate nuclear receptor retinoid X receptor (RXR)/PPARγ signaling by interacting with RXR to modulate adipogenesis. However, whether TBTC affects liver X receptor (LXR)/RXR activity and subsequently the expression of cholesterol mobilizing genes is not known. In this study, we evaluated the ability of TBTC to activate LXR/RXR and ABC transporter A1 (ABCA1) expression. ABCA1 plays a critical role in HDL generation, maintaining cholesterol homeostasis, and cholesterol accumulation-induced diseases, such as atherosclerosis and pancreatic islet dysfunction. In a reporter gene assay, TBTC activated LXRα/RXR but not LXRβ/RXR. In mouse macrophage RAW264 cells, TBTC activated the ABCA1 promoter in an LXR-responsive element dependent manner and increased ABCA1 mRNA expression. TBTC augmented ABCA1 protein levels and apolipoprotein A-I-dependent cellular cholesterol efflux (HDL generation). The LXR-target fatty acid synthase and Spα mRNA levels were also increased by TBTC exposure. We conclude that TBTC has the ability to activate permissive LXRα/RXR signaling and thereby modulate cellular cholesterol efflux.     

Mechanism underlying the olfactory disturbance induced by an intraperitoneal injection of tributyltin chloride in rats

Acute intoxication by tributyltin compounds has been known to induce olfactory disturbances, although the underlying mechanism remains unclear. This study investigates the acute effect of tributyltin chloride (TBTC) on the olfactory bulb in rats. The time-course characteristics of the intra-olfactory concentration of TBTC, the histopathological changes of the olfactory bulb and the olfactory function were examined for 96 h after a single intraperitoneal injection of 2.5 mg/kg of TBTC. The olfactory function was evaluated by the discriminating ability for a cycloheximide solution which has an unpleasant odor for rats. The concentration of TBTC in the olfactory bulb, which was measured using gas chromatography/mass spectrometry, quickly increased to a peak value within 24 h and then decreased. The viable cell number significantly decreased after the TBTC administration in the mitral cell layer and granule cell layer of the olfactory bulb, while apoptotic cells significantly increased in these areas at the same time. Hyposmia was evident 96 h after the TBTC injection, although olfactory testing could not been performed until that time because of anorexia. These results suggest that intraperitoneally injected TBTC was promptly transferred to the olfactory bulb through the blood–brain barrier, induced apoptosis of the cells in the olfactory bulb and finally elicited the olfactory disturbance.     

Bone marrow fibroblasts-conditioned medium regulates the proliferation of leukemic cells

The effect of normal human bone marrow fibroblasts-conditioned medium (BMF-CM) was studied on the proliferation of K562 cells, and on leukemic cells from patients with acute myelocytic leukemia at the time of diagnosis and chronic myelogenous leukemia (CML) at chronic phase. BMF-CM was obtained from normal human bone marrow fibroblasts by using long-term liquid cultures. BMF-CM suppressed the proliferation of K562 cells and leukemic cells from patients with undifferentiated type of leukemia (M1 in FAB classification). However it stimulated the proliferation of leukemic cells from patients with differentiated type of leukemia (M2 in FAB classification), and it slightly stimulated that from patients with CML. These results suggest that normal human BMF regulate the proliferation of leukemic cells in their various stages of differentiation in the bone marrow by releasing a humoral factor.     

Characteristics of bone marrow fibroblasts and their significance in hematopoiesis

Effective hematopoiesis is thought to be maintained by interplaying between the hematopoietic stem cells (HSC) and their supporting stroma which supply an appropriate environment for HSC lodgement, proliferation and differentiation in the bone marrow. Many experiments have made it apparent that the bone marrow microenvironment is essential for normal hematopoiesis. Several recent reports have shown that most human bone marrow adherent cells under long-term liquid cultures are fibroblasts. In this article, the characteristics of the stromal fibroblasts and their role for HSC maintenance and differentiation are described.     

2,3,7,8-tetrachlorodibenzo-p-dioxin does not directly alter the phenotype of maturing B cells in a murine coculture system

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), acting through the aromatic hydrocarbon receptor (AhR), elicits numerous toxicological effects, including immunosuppression. Previous work from our laboratory has suggested that TCDD exposure in mice is associated with altered lymphopoietic development, in particular altered B-cell phenotype in the bone marrow. It remains to be determined which specific hematopoietic populations or subpopulations within the marrow cavity are directly targeted by TCDD. To examine the effects of TCDD on developing B cells in vitro, a stromal coculture model was used. Primary bone marrow cells from male, 6- to 7-week-old C57Bl/6 mice were cocultured separately on two AhR-containing stromal cell lines (M2-10B4 and S17) that support B-cell development in the presence of IL-7. The cocultures were treated with 0 to 10 nM TCDD. Shifts in phenotype were quantified by cell surface marker staining and flow cytometry. Four populations in the maturing B cell (very early pre-pro-B, pre-pro-B, pro-B, and pre-B) were defined for quantification. The results show that the only statistically significant effect of TCDD was within the pre-pro-B population in cultures with the S17 stromal cell line. The increase in number of cells with this phenotype was seen in cultures with both wild-type and AhR-/- primary bone marrow cells. These results suggest that the maturing B220+ B cell is not the direct target for TCDD-induced bone marrow B-cell alterations.     

ASTA Z 7557 (INN mafosfamide) for the in vitro treatment of human leukemic bone marrows

Summary The in vitro treatment of leukemic bone marrows, collected during complete remission, aims at eliminating residual blast cells prior to freezing and preservation, while sparing normal hematopoietic stem cells.We report our experience on the activity of ASTA Z 7557 on human leukemic (CFU-L) and normal hematopoietic stem cells.The sensitivity of human leukemic and normal progenitor cells (CFU-c), detected in semi-solid media cultures, is similar. However, pre-CFUc progenitors detected in long term marrow cultures are much less sensitive to ASTA Z 7557. Therefore autologous bone marrow transplantation can successfully be done with pretreated marrows containing 5±5% residual CFUc. The wide range of stem cells sensitivity to ASTA Z 7557 justify the predetermination of the optimal dose of drug for incubation prior to marrow collection for each individual patient. Our preliminary clinical experience is exposed.     

Tributyltin chloride induces ABCA1 expression and apolipoprotein A-I-mediated cellular cholesterol efflux by activating LXRα/RXR

Organotins, including tri-butyltin chloride (TBTC), are widely used in agricultural and chemical industries and cause persistent and widespread pollution. TBTC has been shown to activate nuclear receptor retinoid X receptor (RXR)/PPARγ signaling by interacting with RXR to modulate adipogenesis. However, whether TBTC affects liver X receptor (LXR)/RXR activity and subsequently the expression of cholesterol mobilizing genes is not known. In this study, we evaluated the ability of TBTC to activate LXR/RXR and ABC transporter A1 (ABCA1) expression. ABCA1 plays a critical role in HDL generation, maintaining cholesterol homeostasis, and cholesterol accumulation-induced diseases, such as atherosclerosis and pancreatic islet dysfunction. In a reporter gene assay, TBTC activated LXRα/RXR but not LXRβ/RXR. In mouse macrophage RAW264 cells, TBTC activated the ABCA1 promoter in an LXR-responsive element dependent manner and increased ABCA1 mRNA expression. TBTC augmented ABCA1 protein levels and apolipoprotein A-I-dependent cellular cholesterol efflux (HDL generation). The LXR-target fatty acid synthase and Spα mRNA levels were also increased by TBTC exposure. We conclude that TBTC has the ability to activate permissive LXRα/RXR signaling and thereby modulate cellular cholesterol efflux.     

Bone marrow toxicity in vitro of chloramphenicol and its metabolites

The effect of chloramphenicol and several of its known and potential metabolites on DNA synthesis of rat and human bone marrow cells was investigated. The nitroso analog of chloramphenicol was the most potent inhibitor of DNA synthesis tested. Its inhibitory effect appeared to be irreversible, while that of chloramphenicol was reversible. The ¹⁴C label of the nitroso analog also bound irreversibly to viable rat bone marrow cells (9.2%) and to heat-inactivated cells (2.6%). In contrast, negligible amounts (0.02%) of the ¹⁴C label of chloramphenicol bound irreversibly to bone marrow cells. These results suggest that the bone marrow toxicity of the nitroso analog of chloramphenicol is related, at least in part, to its marked chemical reactivity.     

Correlation of ionizing irradiation-induced late pulmonary fibrosis with long-term bone marrow culture fibroblast progenitor cell biology in mice homozygous deletion recombinant negative for endothelial cell adhesion molecules

Ionizing irradiation damage to the lung is associated with an acute inflammatory reaction, followed by a latent period and then late effects including predominantly pulmonary fibrosis. The cells mediating fibrosis have recently been shown to derive from the bone marrow hematopoietic microenvironment. Initiation of late pulmonary irradiation lung damage has been correlated with up-regulation of VCAM-1 and ICAM-1 in pulmonary endothelial cells, followed by infiltration of macrophages and bone marrow-derived fibroblasts forming the fibrotic lesions of organizing alveolitis/fibrosis. To determine whether the absence of expression of VCAM-1, ICAM-1, or other adhesion molecules known to be relevant to inflammatory cell attachment to lung endothelial cells was associated with a decrease in irradiation-induced lung fibrosis, homozygous deletion recombinant knockout mice lacking each of several adhesion molecules were tested compared to littermates for survival and development of organizing alveolitis following 20 Gy irradiation to both lungs. Bone marrow culture longevity has been shown to be a parameter, which correlates with both hematopoietic stem cell reserve and the integrity of fibroblast progenitors of the supportive hematopoietic microenvironment; radiation lung survival data were correlated to longevity of hematopoiesis in long-term bone marrow cultures established from tibia and femur bone marrow of the same mice. Homozygous deletion recombinant negative mice including VCAM-1-/-, ICAM-1-/-, E-Selectin-/-, or L-Selectin-/- were irradiated to 20 Gy to both lungs and followed for survival and percent organizing alveolitis at time of death compared to each normal littermate. A significant increase in survival (median 190 days) was detected with L-Selectin-/- compared to littermate control mice (median 140 days) or other groups. Long-term bone marrow cultures from L-Selectin-/- mice showed no detectable difference in marrow fibroblasts or hematopoietic cell biology compared to normal littermates; however, E-Selectin-/- mouse long-term bone marrow cultures showed an increase in total cumulative cell production (1.7 x 10(8) cells per flask) compared to bone marrow cultures from normal littermates (1.8 x 10(6) cells per flask) or other groups. As additional controls, transgenic Sod2 mouse long-term bone marrow cultures and those from HPV16, E6 and E7 cytokeratin 14 transgenic mice were also tested. No detectable difference in hematopoiesis was noted in these cultures compared to littermates. The results suggest a complex pattern of involvement of endothelial specific adhesion molecules and marrow fibroblasts in the cell biologic events associated with late irradiation pulmonary fibrosis.     

六种重组细胞因子对人骨髓造血细胞的体外扩增效应

目的 :观察造血生长因子对骨髓CD34 +细胞的扩增效应及扩增细胞的分化特性。方法 :采用重组人白细胞介素 (rhIL) 1 +rhIL 3+rhIL6 +重组人粒细胞集落刺激因子 +重组人粒 巨噬细胞集落刺激因子和重组人干细胞因子对人骨髓单个核细胞 (MNC)进行刺激 ,动态观察了经上述细胞因子作用后MNC的增殖效应及液态扩增培养后粒 巨噬集落形成率 (CFU GM ) ,利用流式细胞技术 (FACS)分析了扩增前后CD34 +细胞及其亚群的动态变化。结果 :经上述 6种细胞因子作用 2 4d ,骨髓MNC总数是原代MNC的 38.33倍 ,液态扩增 1 0d后 ,CFU GM的数量达高峰 ,是原代MNC的 1 9.0 4倍 ,2 4d时CFU GM总数降至 3.94倍 ,且集落明显小于扩增初期。扩增至 6～ 1 0d ,CD34 +细胞总数是原代MNC的 1 36 .40～ 90 .40倍 ,1 9d时降为 6 1 .40倍。结论 :外源性造血生长因子对CD34 +骨髓细胞具有较强的扩增效应 ,选择适宜的扩增时间是保证骨髓移植后尽快重建造血功能的重要环节。