Action of inhibitor released from neutrophils and leukemic blast cells.

The concept that polymorphonuclear leukocytes, or neutrophils, play a role in feedback control of granulopoiesis has been supported by the finding in bone marrow culture studies that mature neutrophils inhibited formation of granulocytic colonies. The study described in this paper was done to investigate the mechanisms involved. With the use of a modified assay it was found that mature neutrophils released factors that reduced the proliferation of colony-forming cells in cultures stimulated by cell-free colony-stimulating factor. In myeloproliferative and myelodysplastic disorders the amount of inhibitor released by the neutrophils varied greatly. Leukemic blast cells also released inhibitor, and in some cases the amount released per cell was greater than the amount released from normal mature neutrophils. The inhibitory factors released from the neutrophils differed from those previously described in the literature in terms of mode of action and apparent molecular size.     

Studies on control of granulopoiesis in man I. Relationship of leukocyte colony-stimulating activity in vitro to neutrophil count in vivo

We examined the relationship of leukocyte colony-stimulating activity (CSA) in vitro to neutrophil count in vivo. Using a standard two-layer system, cultures of 10⁶ leukocytes were assayed for their ability to stimulate colony formation by human bone marrow colony-forming cells. The total leukocyte CSA per ml (TLCSA) of blood varied directly with the blood neutrophil count in a group of patients with a wide range in blood neutrophil count, and in two patients recovering from neutropenia in whom serial observations were made. In the latter two patients the rise in TLCSA did not antedate the rise in blood neutrophil count, suggesting that blood leukocyte colony-stimulating factor (CSF) per se probably has little biologic significance. However, release into the circulation of cells which generate CSF could be an important way of controlling the amount of CSF acting within the marrow. In one patient the CSA of dialyzed serum increased after the rise in TLCSA, while undialyzed serum contained no CSA.     

Effect of lithium on granulopoiesis in culture.

Lithium carbonate therapy is associated with polymorphonuclear leukocytosis. In vitro studies have shown that lithium ions stimulate formation of granulocytic colonies. In a study undertaken to determine how lithium acts, colony-forming cells uncontaminated by monocytes (which elaborate colony-stimulating factor [CSF] in vitro) were obtained by means of a two-step cell separation procedure. The effects of lithium on colony formation were then studied in (a) cultures stimulated by humoral CSF, (b) cultures in which monocytes were relied upon to synthesize CSF de novo and (c) unstimulated cultures. Lithium enhanced the action of CSF but did not stimulate colony formation in the absence of CSF. In monocyte-stimulated cultures, colony formation increased with lithium concentrations up to 1 mmol/L but this increase paralleled that in CSF-stimulated cultures and therefore was not due to increased CSF production by monocytes. At higher concentrations of lithium, colony formation decreased in the monocyte-stimulated cultures but increased in the CSF-stimulated cultures. A lithium concentration of 4 mmol/L gave the greatest enhancing effect on colony formation in CSF-stimulated cultures and a concentration greater than 1 mmol/L inhibited de novo synthesis of CSF by monocytes.     

Stimulating effect of catechin, an active component of Spatholobus suberectus Dunn, on bioactivity of hematopoietic growth factor

Background Hematopoietic growth factor （HGF） is indispensable to hematopoiesis in the body. The proliferation and differentiation of hematopoietic cells must rely on the existence and stimulation of HGF. This study investigated the effect of catechin, an active component extracted from Spatholobus suberectus Dunn （SSD）, on bioactivity of granulocyte-macrophage colony-stimulating activity （GM-CSA）, burst-promoting activity （BPA） and megakaryocyte colony-stimulating activity （MK-CSA） in spleen condition medium （SPCM） of mice to clarify the hematopoietic mechanism of catechin and SSD.
Methods Spleen cells of mice were separated and spleen condition medium （SPCM） was prepared from spleen cell culture. Bone marrow cells of mice were separated and cultured in a culture system including 10% （v/v） SPCM （induced by catechin in vivo or ex vivo） for 6 days. Granulocyte-macrophage colony forming units （CFU-GM）, erythrocyte burst-colony-forming units （BFU-E） and megakaryocyte colony-forming units （CFU-Meg） formation were employed to assay the effects of different treatment on the bioactivity of GM-CSA, BPA and MK-CSA in SPCM.
Results SPCM induced by 100 mg/L catechin ex vivo could promote the growth of CFU-GM, BFU-E and CFU-Meg, which indicated that catechin could stimulate the production of GM-CSA, BPA and MK-CSA in SPCM. SPCM prepared at the fourth day of spleen cell culture showed the best stimulating activity. The bioactivity of GM-CSA, BPA and MK-CSA in the SPCM prepared after intraperitoneally injecting catechin into mice was also increased. The number of CFU-GM, BFU-E and CFU-Meg gradually increased as the dose of catechin increased and the time of administration prolonged. CFU-GM, BFU-E and CFU-Meg of the high-dose catechin group were significantly higher than those of the control group （P〈0.01） and reached the maximum at the seventh day after administration.
Conclusions This study suggests that catechin extracted from the active acetic ether part of Spatholo     

有机锗对粒-巨噬集落形成细胞的作用

利用体外琼脂半固体培养体系探索了有机锗(Ge-132)对粒—巨噬集落形成细胞(CFC-GM)的作用。结果表明,体系中无集落刺激因子时(CSF),Ge-132不能刺激CFC-GM形成集落。若体系中有CSF,则Ge-132不能增加CFU-GM的集落数量,二者无协同作用。把Ge-132加入到含肺组织的双层培养体系中,则发现它能显著地促进CFC-GM集落的形成,而Ge-132对含其他组织的双层培养体系无此现象。提示Ge-132刺激了肺组织的某些细胞(内皮细胞或者是巨噬细胞),产生活性的集落刺激因子而促进CFU-GM集落的形成。     

五羟色胺促进骨髓基质细胞生长初探

目的:探讨五羟色胺(serotonin,5-HT)对骨髓基质细胞(bone marrow stromal cell,BMSC)体外生长的影响.方法:采用骨髓基质细胞集落培养技术(CFU-F)检测不同浓度5-HT以及5-HT分别与碱性成纤维细胞生长因子(basic-fibroblast growth factor,bFGF),血小板源性生长因子(platelet-derived growth factor,PDGF),血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)对小鼠及人BMSC体外培养的生长促进作用,反转录多聚酶链反应(RT-PCR)检测人BMSC表面5-HT2型受体mRNA表达.结果:5-HT不同浓度(50,100,200,500nM)对小鼠及人BMSC体外培养均有生长促进作用(P＜0.05),显示最大作用浓度为200nM.5-HT可增强bFGF,PDGF和VEGF对BMSC生长促进作用.人BMSC表面有5-HT2A,2B,2C受体mRNA表达.结论:5-HT具有BMSC生长促进作用,并可增强bFGF,PDGF,VEGF促进BMSC的生长,其作用途径可能通过BMSC表面5-HT2型受体,从而促进细胞生长.     

Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophases

Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40 μmol/ L)inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20 μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0.5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.     

Sodium caseinate induces expression and secretion of murine multipotent myeloid cell line 32D macrophage colony-stimulating factor

BACKGROUND: Evidence that sodium caseinate (CasNa) is capable of inhibiting proliferation of hematopoietic precursor cell line 32D and inducing its differentiation into macrophage cells has recently been published. Taking into consideration that hematopoiesis is regulated by growth factors and that macrophage colony-stimulating factor (M-CSF) is a well-known growth factor that induces differentiation of macrophages, in this work we evaluated whether CasNa is capable of inducing expression and secretion of M-CSF in 32D cells. METHODS: We cultured 32D cells in presence and absence of CasNa and compared their proliferation and viability. RNA was extracted from cell lysates to evaluate expression of the gene for M-CSF and its receptor. Cultured conditioned media was used to evaluate presence of M-CSF. RESULTS: Our results showed that CasNa inhibited proliferation of 32D cells and that conditioned media (CM) of these cultures contained M-CSF-like activity. Presence of M-CSF in CM was detected by inhibiting M-CSF activity with anti-M-CSF and presence of this growth factor was confirmed by ELISA assay. We also provided evidence that CasNa induced expression of mRNA for M-CSF in 32D cells as well as increased expression of mRNA for its receptor. CONCLUSIONS: CasNa inhibits proliferation of 32D cells and induces expression of the gene for M-CSF and that of its receptor. It also induces secretion of the bioactive form of M-CSF.     

丹参对外周血中性粒细胞和单核细胞的调整作用

研究丹参对外周血中性粒细胞和单核细胞的化学趋化和超氧化物产物的影响。结果表明：丹参能明显增加两种细胞的超氧化物的产生；并对两种细胞的化学趋化有明显的抑制作用，丹参能调整中性粒细胞的氧化过程，参与化学趋化作用，炎症过程和增强中性粒细胞的杀菌能力。     

双歧杆菌对单核细胞来源的树突状细胞成熟及功能影响的体外研究

目的 研究双歧杆菌对正常成年人外周血单核细胞来源的树突状细胞(dendritic cell, DC)刺激淋巴细胞增殖功能及分泌细胞因子的影响.方法 以GM-CSF、IL-4联合诱导单核细胞生成未成熟DC,加入不同剂量热灭活的双歧杆菌,观察DC形态,检测混合淋巴细胞反应,ELISA法测IL-12、IFN-γ分泌.结果 经双歧杆菌死菌刺激后,可获得具有典型树突状突起形态的DC;诱导后的DC刺激同种异体T淋巴细胞增殖的能力增强(P＜0.01),分泌IL-12、IFN-γ的水平提高(P＜0.05),呈剂量依赖型.结论 双歧杆菌能影响单核细胞来源的DC的分化、成熟及功能的发挥,且不同剂量的双歧杆菌对DC的成熟程度影响有差异.     

蛋白激酶C信号通道对牛肺动脉平滑肌细胞增生的调控作用

探讨蛋白激酶C（PKC）信号通道对体外培养的正常成年牛肺动脉平滑肌细胞（PA SMC）增生的调控作用。结果:PKC活化剂PMA（phorbol 12-myristate 13-acetate）可明显刺激PA SMC增生,PMA为10nmol/L时,3~H胸腺嘧啶掺入率由对照组的（44.9±10.6）min~（-1） /孔增加到（5904.7±607.6）min~（-1）/孔,p<0.01;预先应用PKC抑制剂可阻断PMA的刺激作用,3~H胸腺嘧啶掺入率由（5 117.1±756.2）min~(-1)/孔减少至（90.2±5.3）min~(-1)/孔;3种不同的PKC抑制方法均可部分阻断100ml/L小牛血清对PA SMC增生的刺激作用（均为 P<0.01）;在 PA SMC的全细胞溶解产物中可直接测到PKC酶活性,而经钝化处理后该酶活性消失（P<0.01 ）,与钝化处理可部分阻断血清的PA SMC增生刺激作用相吻合。提示PKC信号通道是PA SMC增生的调控机制之一。     

顽疣净对郎格汉斯细胞刺激T细胞增殖和分泌Th1/Th2细胞因子的影响

目的:观察顽疣净含药血清对对郎格汉斯细胞(LC)刺激T细胞增殖和分泌Th1/Th2细胞因子的影响。方法:以顽疣净含药血清干预分离的人表皮LC。用同种异体混合T淋巴细胞反应,观察顽疣净含药血清对LC刺激T淋巴细胞增殖的影响;用ELISA法检测混合T淋巴细胞反应上清中IFN-γ和IL-10表达,了解顽疣净含药血清对LC调节Th分化的影响。结果:顽疣净含药血清干预后的LC刺激T细胞增殖能力提高,能提高混合T淋巴细胞反应上清中IFN-γ表达,抑制IL-10分泌。结论:顽疣净含药血清体外具有调整LC功能的作用。     

脐血单个核细胞体外扩增的实验研究

目的 :采用集落刺激因子体外扩增脐血单个核细胞 (CBMC) ,研究集落刺激因子对CBMC增殖活性的影响。方法 :用RPMI1 640培养液加入集落刺激因子IL - 3、SCF、GM -CSF及其组合 ,体外扩增CBMC ,健康成人外周血单个核细胞 (PBMC)作为对照组。结果 :加入不同集落刺激因子体外培养CBMC与PBMC后 ,增殖活性均有不同程度的升高 ,以加入IL - 3、SCF、GM -CSF组最为显著 ;CBMC的增殖活性与PBMC比较有显著性差异 (P<0 .0 5)。结论 :集落刺激因子可在体外扩增CBMC ,IL - 3、SCF、GM -CSF之间有明显的协同作用 ;与PBMC相比 ,CBMC有更高的增殖潜能     

CSF延长慢性粒细胞白血病病理克隆体外培养存活期限作用的研究

为观察集落刺激因子（ＣＳＦ）对慢性粒细胞白血病（ＣＭＬ）病理克隆增殖的影响，采用单抗 免疫磁珠二步分离法，从ＣＭＬ患者骨髓标本分离出带有恶性克隆标记的ＣＤ３４＋ＨＬＡ ＤＲ＋细胞，并在体外观察干细胞因子（ＳＣＦ）、白细胞介素 ３（ＩＬ ３）和促红细胞生成素（Ｅｐｏ）联合应用对细胞增殖的影响。结果：ＣＳＦ存在体系与无ＣＳＦ体系相比ＣＭＬ病理克隆消失延迟１４ｄ（Ｐ＜０．０５）。结果提示：ＣＳＦ具有延长ＣＭＬ病理细胞在体外培养存活期限的作用。     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

体外培养恒河猴外周血单核巨噬细胞的研究

目的：建立一种简单、经济、高效地培养恒河猴外周血单核巨噬细胞（monocyte-derived macrophage,MDM）的方法。方法：用肝素钠抗凝管采集健康成年中国恒河猴（Macaca mulatta）全血,密度梯度离心法分离外周血单核细胞（peripheral blood mononuclear cells, PBMCs）。同时用无抗凝剂采血管采集同一只猴外周血,自凝后分离血清。将猴PBMCs置于CELLBIND Surface的96孔（0.8×106个细胞/孔）或48孔培养板（3×106个细胞/孔）中,用含不同百分比的猴自体血清或胎牛血清（fetal calf serum,FCS）的RPMI 1640培养液培养24h后洗弃未贴壁细胞,加入含有猴自体血清或FCS的新鲜培养基继续培养7天后观察细胞形态学。分化良好的猴单核巨噬细胞贴壁能力强,占据板底大部分区域。胞体形态多样,多数呈长梭形。用巨噬细胞标记受体（CD14）抗体染色判断细胞纯度。并用细菌内毒素（LPS）刺激分化的巨噬细胞,检测巨噬细胞炎性因子的表达。此外,用猴艾滋病毒（SIVmac17E-Br、SIVmac251）和人-猴嵌合体艾滋病毒（SHIV KU-1）感染分化良好的猴巨噬细胞,检测病毒在猴巨噬细胞中的复制。结果：在含2%猴自体血清的RPMI 1640培养条件下,大多数（>85%）猴单核细胞能在24h内贴壁,体外分化5-7天后,猴巨噬细胞纯度大于96%。相比而言,含较高浓度（4%,8%或10%）猴自体血清或FCS的RPMI 1640 培养基对猴单核细胞的贴壁和分化作用较差。分化良好的猴巨噬细胞对LPS刺激敏感,可产生多种巨噬细胞炎性因子。此外,这些细胞对SIV或SHIV均易感,产生感染性病毒。结论：含2%猴自体血清的RPMI 1640培养基适于原代猴单核细胞的贴壁和分化。该方法简单、花费少,无需生长因子,且分化效果好,是培养猴艾滋病毒及开展相关免疫学实验的重要手段。     

无细胞胎肝液对大剂量化疗后小鼠骨髓造血细胞的影响

采用小鼠体内液体扩散盒及脾结节法观察了胎鼠和成年小鼠肝脏无细胞液对大剂量环磷酰胺化疗后小鼠骨髓造血细胞增殖与分化的影响。发现无细胞胎肝液能明显促进受抑制骨髓中多能造血干细胞(CFU—S)的集落形成率,但对扩散盒内粒系祖细胞(CFU—D)的增殖分化无促进作用。成年鼠肝无细胞液则对造血细胞的增殖无刺激活性,不能促进化疗后小鼠骨髓中造血干细胞恢复。认为无细胞胎肝液中存在的造血刺激因子能有效缓解细胞毒剂对骨髓多能造血干细胞的抑制。     

重组人血清白蛋白-粒细胞集落刺激因子融合蛋白单次给药对恒河猴造血细胞的动员作用

目的 研究重组人血清白蛋白-粒细胞集落刺激因子融合蛋白(GW003)对造血细胞的动员作用.方法 7只健康成年恒河猴单次皮下注射GW003 150 μg/kg,分别于给药前后不同时间检测外周血细胞数和进行造血细胞集落培养.结果 GW003对健康成年恒河猴造血祖细胞、白细胞、中性粒细胞、未成熟粒细胞及单核细胞均有明显的动员...     

Bovine granulocyte/macrophage and erythroid colony culture: characteristics of the colonies and the assay systems.

Bovine bone marrow granulocyte/macrophage colonies were cultured in vitro in methyl cellulose and in plasma clots using bovine endotoxin-stimulated serum as a source of colony stimulating activity. The endotoxin-stimulated serum was four times as potent as the control serum in the methyl cellulose cultures. No significant increase in the number of colony forming units was observed when bovine marrow cells were maintained in suspension cultures for various periods prior to plating in methyl cellulose. The percentage of glass/plastic adherent cells in bovine marrow cells was observed to be 43% +/- 12 (SD). Benzidine positive erythroid colonies appeared in plasma clot cultures on day 4 and disappeared by day 9. No second population of erythroid colonies appeared either as a function of time or as a function of erythropoietin concentration. The optimum erythropoietin concentration for bovine erythroid cultures was found to be 1.0 unit/mL. A significant difference was observed between animals in their marrow capacity to produce erythroid colonies in culture but no significant difference was observed within individual animals over a period of three months.     

3,6-(二甲氨基)-二苯骈碘杂六环葡萄糖酸盐对AGEP引起的大鼠主动脉平滑肌细胞增殖及牛主动脉内皮细胞内皮素和一氧化氮改变的影响

目的 :观察 3 ,6 (二甲氨基 ) 二苯骈碘杂六环葡萄糖酸盐对AGEP引起的大鼠主动脉平滑肌细胞增殖及牛主动脉内皮细胞内皮素和一氧化氮改变的影响。方法 :采用牛血清白蛋白 (BSA)与不同浓度葡萄糖 (0 ,2 0 ,5 0 ,80mmol·L-1)体外孵育制备糖基化终产物 (AGEP) ,应用 [3 H] TdR掺入法和MTT比色法观察I-93对重度糖化的AGEP诱导的大鼠主动脉平滑肌细胞 (ASMC)增殖的影响 ;应用放射免疫技术及Greiss法观察I -93对AGEP引起的牛主动脉内皮细胞 (BAEC)释放内皮素 1(ET 1)和一氧化氮 (NO)的影响。结果 :I-93 10 -7～ 10 -5mol·L-1能明显抑制AGEP引起的ASMC增殖 ,其 [3 H] TdR掺入量和MTT比色法的最大抑制率分别为79 .4%和 44 .2 %。随AGEP糖浓度的增加 (2 0～ 80mmol·L-1) ,BAEC培养液中ET 1含量亦逐渐上升 [(4 93± 63 )～ (779± 10 5 )ng·L-1] ,I -93 10 -7～ 10 -5mol·L-1能明显抑制重度糖化的AGEP促进ET 1释放的作用 ;I -93对AGEP灭活NO的作用有剂量依赖性抑制效应。结论 :I -93有抑制ASMC增殖的作用 ;对AGEP诱导的BAEC释放ET 1和NO间平衡失调有调节作用 ,在防治阻塞性血管疾病方面I -93具有潜在的应用价值