地塞米松对体外人骨髓基质细胞增殖及成骨分化的影响

目的观察地塞米松对骨髓基质细胞(MSCs)增殖及成骨分化的影响。方法以离心法分离培养MSCs,分别以10~(-9)、10~(-8)和10~(-7) mol/L地塞米松对细胞进行干预,采用四甲基偶氮唑蓝比色法(MTT法)测细胞增殖率；测细胞质碱性磷酸酶(ALP)含量；利用反转录聚合酶链反应(RT-PCR)技术在转录水平检测成骨细胞标记物骨桥蛋白(OPN)mRNA的表达。结果地塞米松干预8 d后,各干预组的吸光度值均低于对照组,10~(-8)和10~(-7) mol/L地塞米松组与对照组相比差异有统计学意义(P＜0．05)；地塞米松干预12 d后,各干预组细胞质ALP含量呈浓度依赖性增加,与对照组相比差异均有统计学意义(P＜0．05)；地塞米松干预12 d后,各干预组均有OPN mRNA表达,且随浓度增加表达增强。结论地塞米松对体外MSCs增殖有抑制作用,但对MSCs成骨分化有促进作用。     

The cell-adhesive effect of basic fibroblast growth factor on pituitary cells in vitro

Both native and recombinant basic fibroblast growth factor (FGF) exerted a marked effect on the morphology of a pituitary clonal somatotroph-like cell line (MtT/S) in vitro. Addition of 10 pg-20 ng bovine basic FGF/ml caused the cells to flatten and spread out over the culture dish, the cells showing strong surface contact. Those in contact with the culture dish were epithelial in appearance, being polygonal, closely apposed and forming a pavement-like monolayer. Basic FGF inhibited cell proliferation in a dose-dependent manner at the higher concentrations tested (0.5-20 ng/ml). This cell-adhesive effect of basic FGF was also observed in normal dispersed pituitary cells. The observed stimulation of pituitary cell adhesion is the first report of a morphological effect of basic FGF on pituitary cells and could explain the physiological significance of basic FGF and its high concentration in the pituitary gland.     

骨髓间充质干细胞的培养及低氧条件对其影响

目的 骨髓间充质干细胞(BMMSC)的体外培养和低氧对于BMMSC增殖作用的影响.方法 成年雄性SD大鼠断颈处死后于75％酒精中浸泡5 min.用全骨髓贴壁法培养细胞,选取生长状态良好的第3代细胞进行鉴定.以单克隆抗体CD45、CD90行流式细胞术鉴定大鼠BMMSC.用四甲基偶氮唑蓝(MTT)法测定第3代BMMSC以及低氧处理的第3代BMMSC的增殖情况.结果 用全骨髓贴壁法分离并培养SD大鼠BMMSC;流式细胞仪检测显示:第3代BMMSC表面特异性标志CD90表达率为96.0％,而非BMMSC表面标志CD45仅为2.5％.MTT结果显示低氧条件下的BMMSC比常氧条件下增殖速率高,并且光镜下观察到细胞状态均一,折光性更好.结论 用全骨髓贴壁法可以在体外分离、培养出纯度较高的SD大鼠来源BMMSC,低氧环境可以刺激BMMSC的增殖.     

The effect of hemopoietic growth factors on the generation of osteoclast-like cells in mouse bone marrow cultures

Multinucleated cells containing tartrate-resistant acid phosphatase were produced in mouse bone marrow cultures in response to 1,25-dihydroxyvitamin D3. These cells resemble osteoclasts in their morphology, possess receptors for calcitonin, and resorb bone in culture. The effects of several hemopoietic regulatory proteins on the generation of these cells were examined in this study. Interleukin-3, granulocyte-macrophage-stimulating factor (GMCSF), and macrophage-stimulating factor strongly inhibited generation of the tartrate-resistant acid phosphatase-containing multinucleated cells with approximate EC50 values of 3, 6, and 3 colony-forming units/ml, respectively. Granulocyte colony stimulating factor, interleukin-6, and leukemia inhibitory factor had no effect on the generation of these cells. In addition, we observed that the number of these cells was reduced when the bone marrow was plated at high cell density, and that this inhibitory effect was reversed by the addition of neutralizing antibodies directed against GMCSF. These findings suggest that GMCSF and other hemopoietic factors secreted by cells in the bone marrow regulate development of the osteoclast-like cells, possibly by diverting common precursor cells to alternate pathways.     

不同浓度甲氨蝶呤对外周血单个核细胞表达和分泌白细胞介素-17的影响

目的 体外观察不同浓度的甲氨蝶呤(MTX)对外周血单个核细胞(PBMCs)表达和分泌白细胞介素(IL)-17的影响,探究MTX有效治疗类风湿关节炎(RA)的可能机制.方法 分离RA患者和健康人PBMCs,予不同浓度MTX预处理,抗人CD3和抗人CD28刺激后,37 ℃体积分数为0.05的CO2培养箱培养.分别于培养的不同阶段收集细胞或上清液,采用反转录聚合酶链反应(RT-PCR)技术分析IL-17mRNA的表达水平;酶联免疫吸附试验(ELISA)法检测细胞培养液中IL-17的浓度;荧光抗体标记细胞表面抗原CD4和胞内蛋白 IL-17,流式细胞仪分析CD4+IL-17+细胞的比例.结果 比较采用两两配对t检验或独立样本t检验.结果 浓度为0.1、1.0、5.0、25.0μg/ml MTX组IL-17mRNA/GAPDH比值分别为(0.58±0.09、0.48±0.11、0.50±0.09、0.51±0.14),与无药抗体组(0.76±0.08)组间两两比较差异有统计学意义(P＜0.01);IL-17分泌水平则分别为(121±54)、(104±45)、(90±36)、(115±41)pg/ml,与无药抗体组(370±187)pg/ml组间两两比较差异有统计学意义(P＜0.01);MTX处理组CD4+IL-17+细胞比例的平均值下降,但统计学检验差异无统计学意义(P＜0.05).结论 MTX可以有效抑制PBMCs合成和分泌IL-17,而且在一定的剂量下可发挥显著作用,加大剂量并不能增强该作用.     

人巨细胞病毒对粒-单及红系祖细胞体外增殖的影响

目的：探讨人巨细胞病毒(HCMV)对脐血粒—单系祖细胞(CFU—GM)、红系爆式祖细胞(BFU—E)及红系祖细胞(CFU—E)体外增殖的抑制作用。方法：采用造血祖细胞体外半固体培养技术，研究HCMV—ADl69株对脐血CFU—GM、BFU—E及CFU—E集落生长的影响；用聚合酶链反应(PCR)技术检测集落细胞中HCMV—ADl69 DNA。结果：①HCMV—ADl69对CFU—GM、BFU—E及CFU—E均有抑制作用。②其抑制作用与HCMV浓度有关，高浓度(1：10，1：100)感染组与对照组比较集落产率明显下降(P＜0.01)，集落维持的时间明显缩短(P＜0.01)，而低浓度组(1：1000)无此作用。③经PCR检测发现病毒感染组CFU—GM、BFU—E及CFU—E集落细胞中有HCMV—ADl69 DNA存在。结论：①HCMV—ADl69可抑制CFU—GM、BFU—E及CFU—E集落的生长。②造血祖细胞是HCMV—ADl69的宿主细胞之一，HCMV—ADl69能直接感染造血祖细胞。HCMV感染患儿出现的粒细胞减少和贫血可能与此有关。     

The effect of cytomegalovirus on hemopoiesis: in vitro evidence for selective infection of marrow stromal cells

We studied the effects of adding cytomegalovirus (CMV) in vitro to normal human bone marrow mononuclear cells (BM-MNCs), committed myeloid progenitor cells, primitive myeloid blast-colony forming cells, and pre-formed marrow stromal cell monolayers in order to shed light on the mechanism by which hemopoiesis is suppressed in patients who acquire systemic CMV infection after allogeneic bone marrow transplantation. Incubation of BM-MNCs or committed progenitor cells with laboratory strain AD169 or wild strain CMV had no significant effect on total colony numbers or the morphology of component cells. CMV mRNA was not identified by in situ hybridization. In contrast, incubating marrow stromal monolayers with CMV produced specific cytopathic effects in fibroblasts and adipocytes and reduced the capacity of the stromal layers to support the proliferation of primitive myeloid progenitor cells. We conclude that CMV infection may impair hemopoiesis in vivo by a direct effect on the cellular components of the marrow stroma.     

The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro

This study compares the effects of vitamins 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 on populations of chondrocytes at different developmental stages. Confluent third passage chondrocytes derived from the resting zone and adjacent growth region of rat costochondral cartilage were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and increasing concentrations of hormone. After determination of cell number, matrix vesicles and plasma membranes were isolated by differential centrifugation. The effects of hormone on alkaline phosphatase, 5'-nucleotidase, ouabain-sensitive Na+/K+-ATPase, and phospholipid composition were dependent on vitamin D metabolite and were cell specific. Growth cartilage chondrocytes responded primarily to 1,25-(OH)2D3, whereas resting zone cells responded primarily to 24,25-(OH)2D3. 1,25-(OH)2D3 inhibited growth cartilage cell number at pharmacological concentrations and had no effect on resting cartilage cell number. In contrast, 24,25-(OH)2D3 appeared to stimulate resting cartilage cell number at physiological concentrations and inhibit these cells at pharmacological doses, but had no effect on growth cartilage chondrocytes. These data were supported by [3H]thymidine incorporation studies. 1,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase activity, and Na+/K+-ATPase activity in the matrix vesicles of growth cartilage cells. 1,25-(OH)2D3 also stimulated Na+/K+-ATPase activity in the matrix vesicles and plasma membranes of resting zone cells. Incubation with 24,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase, and Na+/K+-ATPase in the matrix vesicles produced by resting zone cells. In addition, 24,25-(OH)2D3 stimulated Na+/K+-ATPase activity in the plasma membranes of resting zone cells as well as in both matrix vesicles and plasma membranes of growth cartilage cells.     

Spleen stromal cells support haemopoiesis and in vitro growth of dendritic cells from bone marrow

Long-term stroma-dependent cultures from murine spleen have been previously shown to support dendritic cell (DC) development in vitro. Secondary cultures have now been established using a splenic stromal cell layer overlaid with cells from different lymphoid sites. Cells resembling DCs can be generated in vitro from unfractionated murine lymphoid cells in the absence of added growth factors. Bone marrow (BM) cultures are the most successful but some cultures have been derived from spleen and thymus. Large numbers of mononuclear cells with dendritic morphology can be generated from BM within 2 weeks and cell production in many cultures has been maintained for at least 6 months. A significant proportion of cells binds antibodies specific for DC markers. No lymphoid cells, mast cells or granulocytes are detectable in culture by antibody and histochemical staining and light and electron microscopy. As with cells generated in primary cultures, cells grown in secondary cultures are equally potent stimulators of both syngeneic and allogeneic mixed lymphocyte reactions, confirming their function as antigen-presenting cells. They are also capable of endocytosing and presenting protein antigen to the D10.G4.1 Th2 clone and to unprimed T cells. This study confirmed the presence of DC precursors in multiple lymphoid sites which can be induced to proliferate in the presence of a spleen stromal cell monolayer. The secondary culture system provides an ideal in vitro model for investigation of the development of DC from different tissue sites. It also provides a stable and continuous resource of cells for further studies on DC development.     

The effect of growth factors in fracture healing and composite grafts of growth factors and marrow mesenchymal cells

Growth factors have been reported to show important roles in fracture healing process. Many studies of experimental animal models demonstrated that some growth factors could enhance bone formation. Clinical applications using the growth factors have been reported, however, effective strategies have not been established. Here, we report a mini review of the growth factors and our new technology, which utilizes growth factors and marrow mesenchymal stem cells.     

The role of fibroblastoid cells and macrophages from mouse bone marrow in the in vitro growth promotion of haemopoietic tumour cells.

Adherent cells from mouse bone marrow have been shown to promote the in vitro growth of the AVRij-1 tumour cell line. The experiments presented here suggest that the adherent cells involved in this phenomenon are the progeny of bone marrow derived fibroblastoid colony forming units. The latter were characterized by means of cell density distribution analysis. They had a broad distribution pattern and an average peak cell density of 1.069 g.cm-3. The growth promotion activity exerted by adherent cell layers from the various density fractions on the AVRij-1 tumour cell line coincided with the distribution of fibroblastoid colony forming units. On the other hand, the presence of macrophages in the adherent layers seemed to be non-essential for the in vitro promotion of growth of the AVRij-1 cell line.     

Inhibitory effect of norcantharidin on the growth of human gallbladder carcinoma GBC-SD cells in vitro

Introduction P rimary gallbladder carcinoma is a lethal and aggressive malignant neoplasm with a dormant course, difficult diagnosis, early metastasis or recurrence, and poor prognosis.[1-3] The only potentially curative therapy for gallbladder carcinoma is surgical resection. Unfortunately, most patients with this type of cancer present with advanced andunresectable disease-only 10%-30% of patients in culture medium overnight, they were treated with can be considered for surgery on presentati…     

The effects of vitamin D metabolites on phospholipase A2 activity of growth zone and resting zone cartilage cells in vitro

Third passage confluent cultures of cartilage cells, initially derived from the growth zone (GC) and resting zone (RC) of rat costochondral cartilage, were incubated with either 10(-11)-10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 10(-9)-10(-6) M 24,25-(OH)2D3. Plasma membranes and extracellular matrix vesicles were isolated, and specific activities of phospholipase A2 and alkaline phosphatase were determined. The results demonstrate that the response to hormone is both cell and membrane specific. 1,25-(OH)2D3 produces an increase in GC matrix vesicle alkaline phosphatase and phospholipase A2 specific activities at 10(-9) and 10(-8) M, but has no effect on these enzyme activities in RC membranes. RC cultured in 24,25-(OH)2D3 exhibit increased matrix vesicle alkaline phosphatase but decreased phospholipase A2 activities at 10(-7) and 10(-6) M hormone. No effect on the RC plasma membrane enzymes or on GC plasma membrane or matrix vesicle enzymes was observed. The data suggest that changes in membrane fluidity due to phospholipase A2 activity may play a role in regulating alkaline phosphatase activity in response to vitamin D metabolites and that this regulation in GC and RC may proceed by different mechanisms.     

Studies on bone marrow in vitro; the effect of anoxia and hyperoxia on explanted bone marrow

The effect of various oxygen tensions on explanted bone marrow fragments wasstudied. It was found that gas mixtures containing 1, 3, 5, 10 and 12 per cent oxygenhave an injurious effect on hemic cells. Bone marrow maintained in these gas mixtures showed various degrees of degeneration, which was the more pronounced thelower the oxygen tension. Mitotic activity was also found to be reduced under theinfluence of low oxygen tension.

Bone marrow cultures maintained in a gas mixture containing 15 per cent oxygendid not show appreciable changes and were similar to the controls.

Increased rate of maturation and multiplication occurred in bone marrow cultures maintained in an excess of oxygen, i.e. 50 per cent.

The significance of these findings in the light of observations on the effect ofanoxia in vivo has been discussed, and reported findings on the effect of low oxygentensions on other tissues in vitro have been briefly reviewed.

     

干扰素α对真性红细胞增多症患者非促红细胞生成素依赖性红系祖细胞生长的影响

目的:探讨干扰素α(IFN-α)治疗真性红细胞增多症(PV)的作用机制。方法:采用体外半固体集落培养的方法,观察IFN-α对PV患者体内外骨髓非促红细胞生成素(Epo)依赖性红系爆式集落形成单位(BFU-E)集落生长的影响。结果:28例PV患者中有26例骨髓中存在非Epo依赖性BFU-E集落生长,平均集落数为196±15/2×10~5单个核细胞(MNC),对照组均无非Epo依赖性BFU-E集落生长。IFN-α在体外对PV患者骨髓中非Epo依赖性BFU-E集落生长呈剂量依赖性抑制作用;IFN-α治疗后,PV患者骨髓非Epo依赖性BFU-E集落数与治疗前比较明显减少(P<0.05),治疗时间越长,BFU-E集落数减少越明显。结论:IFN-α在体内外对PV患者骨髓非Epo依赖性BFU-E集落生长有抑制作用。     

The effect of lithium on growth factor production in long-term bone marrow cultures

We have previously reported that lithium chloride (LiCl) stimulates the production of granulocyte-macrophage colony-forming cells (GM-CFC), pluripotent stem cells (CFU-S), and differentiated granulocytes, macrophages and megakaryocytes in murine Dexter marrow cultures and that this effect appears to be mediated indirectly by a radioresistant adherent marrow cell. In this study we have established that exposure of murine Dexter cultures to LiCl (4 mEq/L) causes an increase of colony-forming cell megakaryocytes (CFU-meg) over 1 to 6 weeks of culture in both supernatant (188% to 611%) and stromal phases (123% to 246%). Moreover, we have shown that lithium treatment of either irradiated (1,100 rad) or unirradiated stromal cells increased production of activities stimulating formation of megakaryocyte, granulocyte, macrophage, and mixed lineage colonies and proliferation of the factor-dependent cell line, FDC-P1. This FDC-P1 stimulatory activity was completely blocked by an antibody to purified recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF). The baseline or lithium-induced--stromal-derived bone marrow colony stimulating activity was partially blocked by the antibody to rGM-CSF and by an antibody to purified colony stimulating factor I (CSF-1); the two antibodies combined resulted in greater than 90% inhibition of the lithium-induced marrow stimulatory activity. In addition, radioimmunoassay (RIA) showed that although CSF-1 was detectable in supernatants of these cultures, exposure to lithium did not increase CSF-1 levels. These data indicate that Dexter stromal cells produce CSF- 1 and GM-CSF and that lithium appears to exert its stimulatory effects on in vitro myelopoiesis by inducing production of GM-CSF.     

The effect of bone marrow transplantation on the osteoblastic differentiation of human bone marrow stromal cells.

Osteoporosis is a serious and relatively common complication of transplantation procedures. However, little is known about the exact mechanism or severity of osteoporosis complicated by bone marrow transplantation (BMT). We conducted both ex vivo and clinical studies to identify the mechanism and extent of bone loss after BMT. In a prospective clinical study, we intended to identify the changes in bone turnover markers and bone mineral density (BMD) after BMT. During a 1-yr follow-up, BMD was measured before BMT and 1 yr after BMT in 67 patients undergoing BMT. Biochemical markers of bone formation and resorption were measured in all patients at short-term intervals during the yearlong follow-up. In ex vivo study, we cultured human bone marrow cells of normal controls and BMT recipients in osteogenic medium and compared their osteogenic potential. Using a DNA fingerprinting method, we also investigated the origin of bone marrow stromal cells that were harvested 3-4 wk after BMT. In a clinical study of 67 patients undergoing BMT, the mean serum carboxy-terminal cross-linked telopeptide of type I collagen increased progressively until 4 wk after BMT. Thereafter, it began to decrease and reached basal values after 1 yr. Serum osteocalcin decreased progressively until 3 wk after BMT and reached basal values after 3 months. One year after BMT, lumbar spine BMD had decreased by 3.3% (P < 0.05), and total proximal femoral BMD had decreased by 8.9% (P < 0.001). For the ex vivo study, bone marrow was obtained from healthy donors (n = 7) and transplant recipients (n = 7). Then, mononuclear cells including marrow stromal cells were isolated and cultured to osteoblastic lineage. Alkaline phosphatase activities of each group were measured by the time course of secondary culture, and the mineralizing potentials were compared between the two groups. Cells cultured in our system showed characteristics of osteoblast-like cells differentiated from marrow stromal cells. They were initially in a fibroblastic-like spindle shape and became cuboidal with the formation of nodules that were later confluent. The cells were stained to both alkaline phosphatase histochemistry and Von Kossa histochemistry, demonstrating that these cells were of osteoblastic lineage differentiating from marrow stromal cells. The mean time required for the near-confluence in the primary culture was 15 and 22.9 d in healthy donors and transplant recipients, respectively (P = 0.003). Alkaline phosphatase activity was significantly lower in the bone marrow recipients than in the healthy donors at d 7 and 10 of the secondary cultures. The period at which peak activity of alkaline phosphatase was reached was also delayed in the osteoblasts derived from BMT recipient bone marrow compared with those of healthy donors. Using Von Kossa histochemistry, much more mineralization was observed in osteoblasts of healthy donors than those of BMT recipients. After BMT, although the peripheral mononuclear cells in the recipients were of donor origin, the bone marrow stromal cells were of recipient origin according to the PCR analysis using YNZ 22 mini-satellite probe. In conclusion, the differentiation of bone marrow stromal cells into osteoblasts was impaired after BMT, and this might contribute to post-BMT bone loss.