小鼠骨髓内皮细胞分泌的细胞因子对造血干/祖细胞增殖的影响

应用小鼠骨髓内皮细胞株细胞传代培养,收集无血清条件培养液（ｍＢＭＥＣ－ＣＭ）,经超滤分成大于１０ｋＤ和小于１０ｋＤ两组分,分别观察两组分的ｍＢＭＥＣ－ＣＭ对小鼠骨髓造血干／祖细胞ＣＦＵ－ＧＭ,ＨＰＰ－ＣＦＣ,ＣＦＵ－Ｅ,ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ的影响。结果表明：含分子量大于１０ｋＤ物质的ｍＢＭＥＣ－ＣＭ的保留液能明显刺激ＣＦＵ－ＧＭ,ＨＰＰ－ＣＦＣ,ＣＦＵ－Ｅ,ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ生长;     

骨髓内皮细胞产物对CFU-F、CFU-GM和WEHI-3细胞生长的作用

为了研究骨髓内皮细胞的造血调控功能,采用串联超波技术脞小鼠骨髓内皮细胞无血清条件培养液（ｍＢＭＥＣ－ＣＭ）中制血出不同相对分子质量（Ｍｇｔ）范围的超滤组分,进行细胞集落形成实难,检测了ｍＢＭＥＣ－ＣＭ及其超滤组分对骨髓成纤维祖细胞（ＣＦＵ－Ｆ）、粒巨噬系造血祖细胞（ＣＦＵ－ＧＭ）和小鼠业单系白现细胞系ＷＥＨＩ－３细胞生长的作用。结果显示：ｍＢＭＥＣ－ＣＭ抑制ＣＦＵ－Ｆ的生长,对ＣＦＵ－ＧＭ和ＷＥＨ     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖…

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１,ＩＬ－６,ＷＥＨＩ３条件培养液及Ｌ９２９条件培养液所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖的影响

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞（ＨＰＰ－ＣＦＣ）和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１、ＩＬ－６、ＷＥＨＩ３条件培养液（ＷＥＨＩ３－ＣＭ,含有ＩＬ－３）及Ｌ９２９条件培养液（Ｌ９２９－ＣＭ,含有Ｍ－ＣＳＦ）所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。这些结果提示ＬｉＣｌ对ＨＰＰ－ＣＦＣ和ＣＦＵ－ＧＭ的作用不同,可能锂有诱导ＨＰＰ－ＣＦＣ向成熟细胞分化的作用     

脐带血清在骨髓造血祖细胞培养中的效应

目的：探讨人脐带清（ＣＢＳ）在骨髓造血祖细胞培养中的效应。方法：用人骨髓细胞进行ＣＦＵ、ＧＭ、ＶＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－ＧＥＭＭ培养。结果：ＣＢＳ能直接刺激骨髓细胞ＣＥＵ－ＧＭ的形成。与血型相同害无关。四人份以上的混合ＣＢＳ（ＭＣＢＳ）,刺激活性高且稳定。１０％ＭＣＢＳ相当于６５．６μｇ／Ｌ ＧＭ－ＣＳＦ、０．２３、０．３、０。．４６ｋＵ／Ｌ ＥｐＯ对ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、Ｃ     

低分子量硫酸葡聚糖对小鼠造血干细胞动员作用的研究

给小鼠静脉注射低分子量（＜１０￣４ｕ）硫酸葡聚糖（ＤＳ）１５ｍｇ／ｋｇ后外周血中白细胞、单个核细胞（ｍｏｎｏｎｕｃｌｅａｒｃｅｋ,ＭＮＣ）、ＣＦＵ－ＧＭ、ＢＦＵ－Ｅ和ＣＦＵ－Ｍｉｘ产率等指标出现时相性变化。给药后１ｈ开始升高,２ｈ达到高峰、分别为药前值的２．２、２．６、３．８、４．４和３．０倍,７ｈ时趋向正常。给药后２ｈ上述各类细胞在外周血中的含量随着ＤＳ的剂量增加而增加。白细胞、ＭＮＣ计数在ＤＳ１８０ｍｇ／ｋｇ时达到峰值,均为对照组的４倍。２４０ｍｇ／ｋ８时未见明显增加。不同剂量ＤＳ对各系祖细胞均有不同程度的动员作用,ＤＳ剂量１５－３０ｍｇ／ｋｇ效果最好,每升血中ＣＦＵ－ＧＭ、ＢＦＵ－Ｅ、ＣＦＵ－Ｍｉｘ的数量分别相当于对照组的５．０、１１．９和８．８倍。其峰值出现时间与白细胞、ＭＮＣ不同,表明ＤＳ对不同类型细胞的作用机制也不尽一致。经口给小鼠投以ＤＳ２４０和４８ｏｍｇ／ｋｇ后,未见外周血中白细胞、ＭＮＣ计数有显著性升高,提示对造血干细胞没有动员作用。     

SOD对骨髓造血祖细胞在4°C条件下保存损伤的防护作用

为了探讨抗氧化剂对造血干细胞在低温条件下损伤的防护作用,将小鼠骨髓细胞置于４℃条件下保存,观察在保养液中加入不同浓度的Ｃｕ、Ｚｎ、ＳＯＤ对细胞死伤的防护效果和马血清对细胞回收率的影响。结果表明,含２０％的马血清保养液中加入ＳＯＤ１．６５Ｕ或０．１６５Ｕ／ｍｌ,保存３天,ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ、ＢＦＵ－Ｅ、ＣＦＵ－Ｍｅｇ、ＣＦＵ－Ｍｉｘ的产率分别为对照组（不加ＳＯＤ）的６．２、２．６、２．９、４．０和５．１倍,明显提高了造血祖细胞的活存率,其中ＣＦＵ－ＧＭ和ＣＦＵ－Ｍｉｘ达到保存前水平。ＳＯＤ的有效作用机理不是对造血祖细胞增殖调控作用,而是防护了细胞的死亡。这可能与其清除过氧自由基的抗氧化作用有密切关系。     

SOD对骨髓造血祖细胞在4℃条件下保存损伤的防护作用

为了探讨抗氧化剂对造血干细胞在低温条件下损伤的防护作用,将小鼠骨髓细胞置于４℃条件下保存,观察在保养液中加入不同浓度的Ｃｕ、Ｚｎ、ＳＯＤ对细胞死务的防护效果和马血清对细胞回收率的影响。结果表明,含２０％的马血清保养液中加入ＳＯＤ１．６５Ｕ或０．１６５Ｕ／ｍｌ,保存３天,ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ、ＣＦＵ－Ｍｅｇ、ＣＦＵ－Ｍｉｘ的产率分别为对照组的６．２、２．６、２．９、４．０和５．１倍。明显提高了造     

造血生长因子结构与功能的相关性研究

概述了多系（ｍｕｌｔｉ）－集落刺激因子（ＣＳＦ）中性粒系－巨噬系（ＧＭ）－ＣＳＦ,Ｇ－ＣＳＦ,Ｍ－ＣＳＦ,红细胞生成素,白介素－１、２、５、６等９种造血生长因子结构－功能相关性研究的主要进展。     

脑肿瘤抑制因子的功能研究

本文证明了胎脑组织中存在一类可以抑制人白血病细胞系的生长的低分子天然抑癌物．这种抑瘤物抑制活性分布在小于１０ｋＤａ组分,经Ｓｅｐｈａｄｅｘ－Ｇ２５凝胶过滤可分离为单一组分,且具有广谱的抗肿瘤效应．体外可抑制人白血病、肝癌、胃癌细胞系和小鼠粒、单核和淋巴系白血病细胞,而对人骨髓ＣＦＵ－ＧＭ和小鼠骨髓ＣＦＵ—ＧＭ的抑制作用较弱。     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Expression of hematopoietic inhibitory factors in mouse fibroblasts,macrophages and endothelial cells

Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT-PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10. The retentate contained substances whose molecular weights were >10 kD, whilst the filtrate contained substances with molecular weights <10 kD. The effect of conditioned media and their components on colony forming unit-granulocyte-macrophage (CFU-GM) were investigated. The results showed: (1) six cytokines, MIP-1alpha, MIP-2, TGF-beta, TNF-alpha, IFN-gamma and Tbeta(4), inhibited the growth of CFU-GM when murine WEHI-3 conditioned medium was added to the culture system as a source of colony stimulation. (2) The original endothelial cell conditioned medium (E-CM) did not affect the production of CFU-GM, but the >10 kD component of E-CM increased its production, and the <10 kD component decreased it. Both fibroblast conditioned medium (F-CM) and the >10 kD component of F-CM stimulated proliferation of CFU-GM, but the <10 kD component suppressed it. All three components of macrophage conditioned medium (M-CM) inhibited the growth of CFU-GM. (3) Expression of four of the mRNAs, namely MIP-2, TNF-alpha, INF-gamma and Tbeta(4), was seen in all three types of stromal cells, while TGF-beta mRNA was only seen in endothelial cells and macrophages, and MIP-1alpha mRNA in endothelial cells and fibroblasts. The inhibitors TGF-beta, MIP-1alpha, and Tbeta(4)have an inhibitory effect on the growth of CFU-GM, but TNF-alpha, INF-gamma and MIP-2 do not.     

小鼠骨髓内皮细胞条件培养液复合FL及TPO对HPP—CFC及CFU—GM增殖的影响

通过传代培养小鼠骨髓内皮细胞,收集无血清条件培养液（ECM）,并经超滤得到大于10kD的浓缩液,分别观察ECM和大于10kD的浓缩液复合flt3ligand(FL)及thrombopoietin(TPO)对体外培养HPP-CFC、CFU-GM的影响,结果表明：ECM或大于10kD的浓缩液对HPP-CFC、CFU-GM的生长均有支持作用;FL或/和TPO与ECM或大于10kD的浓缩液合用能加强对HPP-CFC、CFU-GM生长的刺激作用;FL加TPO与ECM或大于10kD的浓缩液合用对HPP-CFC、CFU-GM生长的刺激作用更加明显;选择FL和TPO特异性的引物,用RT-PCR技术未能检测到小鼠骨髓内皮细胞有FL和TPOmRNA的表达。     

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.     

小鼠LAK细胞对粒—单系祖细胞增殖的影响

Murine lymphokine-activated killer (LAK) cells were generated from spleen cells of C57/BL6 mice by culture of spleen cells in vitro for 72 hours in medium containing 500 units/ml recombinant human interleukin 2 (IL-2), and effects of these LAK cells on proliferation of syngenic myeloid progenitor cells (CFU-GM) were observed. After 3 days culture, LAK cells were assayed for their cytotoxicity in a 4 hours 51Cr-release test. Either natural killer (NK) cell sensitive YAC-1 lymphoma cells or NK cell resistant LP-3 and WEHI-164 fibrosarcoma cells were efficiently lysed by murine LAK cells. When LAK cells were added into culture system in a final concentration of 5 x 10(4)/ml, 2 x 10(5)/ml, 8 x 10(5)/ml, CFU-GM were increased by 55.2%, 165.5%, and 194.4% of control respectively. LAK-CM also showed augmentative effect on CFU-GM growth. When 10% (v/v) of LAK-CM were added into culture system, CFU-GM were increased by 51.4% of control, but LAK-CM alone could not stimulate CFU-GM growth. Again, effects of LAK-BMC interaction on CFU-GM formation were investigated. CFU-GM were inhibited to 27.6% of control when 1 x 10(5) BMC were mixed with 8 x 10(5) LAK cells and incubated for 4 hours prior to CFU-GM culture. These data suggest that (1) LAK cells may secrete co-CSF which showed synergistic effect with CSF on CFU-GM proliferation: (2) When LAK cells contact with BMC, they showed significant cytotoxicity to myeloid progenitor cells which mediated decrease of CFU-GM formation.     

Effects of lymphokines and immune complexes on murine placental cell growth in vitro

Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation. Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells. The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone). Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol). Addition of pseudo "immune complexes" in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium. In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation. The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations. On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

γ-Tocotrienol inhibits angiogenesis of human umbilical vein endothelial cell induced by cancer cell

Antiangiogenic therapy mediated by food components is an established strategy for cancer chemoprevention. Growth factors play critical roles in tumor angiogenesis. A conditioned medium containing growth factors from human gastric adenocarcinoma SGC-7901 cell conditioned medium was used as an angiogenic stimulus in this study. The purpose of this study was to evaluate the inhibitory effect and possible mechanism of γ-tocotrienol on tumor angiogenesis. The results showed that γ-tocotrienol (10-40 μmol/L) significantly suppressed proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by SGC-7901 cell conditioned medium in a dose-dependent manner. γ-Tocotrienol (800-1200 μg/egg) also inhibited new blood vessel formation on the growing chick embryo chorioallantoic membrane in a dose-dependent manner. Moreover, the inhibitory effects of γ-tocotrienol on HUVECs were correlated with inducing the apoptosis and arresting cell cycle at the G₀/G₁ phase at a dose of 40 μmol/L γ-tocotrienol. In addition, γ-tocotrienol inhibited angiogenesis in HUVECs by down-regulation of β-catenin, cyclin D1, CD44, phospho-VEGFR-2 and MMP-9. The antiangiogenic effects of γ-tocotrienol on HUVECs may be attributable to regulation of Wnt signaling by decreasing β-catenin expression. Thus, our results suggest that γ-tocotrienol has a potential chemopreventive agent via antiangiogenesis.     

Endothelial heparan sulfate is necessary but not sufficient for control of vascular smooth muscle cell growth

The state of the endothelial cell (EC) determines the nature of its control of vascular smooth muscle cell (vSMC) biology. Conditioned medium from postconfluent ECs inhibits vSMC proliferation, whereas subconfluent conditioned medium from the same ECs has a stimulatory effect. We and others have identified confluent endothelial cells' production of heparan sulfate proteoglycans (HSPG) as critical to vSMC growth control. The question that arises is whether the stimulation that is observed with subconfluent cells is from (1) aberrant HSPG production, (2) elaboration of noninhibitory species of HSPG, or (3) production of other factors, such as mitogens, which counteract the inhibitory HSPG to stimulate vSMCs. We studied the relative effects of conditioned medium produced by both subconfluent and postconfluent EC cultures on vSMC growth. Conditioned medium was fractionated into nonproteoglycan (non-PG) and proteoglycan (PG) components by anion-exchange chromatography. The PG fractionation profile and the antiproliferative activity of the HSPGs isolated from both subconfluent and postconfluent EC-conditioned media were similar. However, the HSPG fraction alone could not approach the inhibitory potential of unfractionated conditioned medium from postconfluent EC cultures. Non-PG proteins produced by the endothelial cultures had no effect on vSMC growth on their own. Yet, when they were mixed together with HSPG fractions, from either subconfluent or postconfluent EC cultures, the full growth effects were returned. Non-PG protein fractions from postconfluent cultures with HSPG fractions gave maximal inhibition of vSMC growth, whereas non-PG protein fractions from subconfluent EC cultures with HSPG fractions produced the maximal stimulation. Thus, whereas the net stimulatory or inhibitory effect on vSMC growth of EC-conditioned medium is density dependent, this effect does not result from a difference in the antiproliferative heparan sulfate component but rather from non-PG proteins that interact with the heparan sulfates.