人参总皂甙对造血生长因子活性及其mRNA表达的影响

目的　探讨人参“补气生血”的现代生物学机理。　方法　采用核酸分子原位杂交和造血生长因子生物活性检测等技术,研究人参总皂甙（ＴＳＰＧ）对造血生长因子活性及其ｍ ＲＮＡ表达的影响。　结果　ＴＳＰＧ能显著促进小鼠骨髓基质细胞（ＢＭＳＣ）、腹腔巨噬细胞（ＰＭ）和脾细胞（ＳＰＣ）的粒－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）及白细胞介素－６（ＩＬ－６）ｍ ＲＮＡ的表达,经ＴＳＰＧ诱导制备的ＢＭＳＣ、ＰＭ和ＳＰＣ条件培养上清液含有较高的爆式红系集落刺激活性（ＢＰＡ）、粒－巨噬细胞集落刺激活性（ＧＭ－ＣＳＡ）和巨核细胞集落刺激活性（ＭＫ－ＣＳＡ）。　结论　ＴＳＰＧ促进血细胞生成的机理与其诱导造血生长因子的基因表达有密切关系。     

造血细胞集落刺激因子检测的研究进展

造血细胞集落刺激因子检测方法已由经典的半固体培养法逐步被免疫学法和依赖细胞学检测法所取代，形成了ＥＬＩＳＡ和依赖细胞系ＭＴＴ比色分析法。本文比较了经典的生物学法和免疫学法的优缺点。     

M—CSF诱导MnSOD表达减轻RAW264.7细胞氧化损伤

为探讨巨噬细胞集落刺激因子（M－CSF）对单核巨噬细胞氧化损伤的保护作用,以富含M－CSF的小鼠L929细胞条件培养液（L929－CM）作为M－CSF来源,以小鼠巨噬细胞系RAW264．7为模型,从形态学上观察了M－CSF对叔丁基氢过氧化物（tbOOH)引起的RAW264。7细胞氧化损伤的保护作用;并采用酶活性测定、TR－PCR等方法,他M－CSF对RAW264．7细胞超氧化物歧化酶（SOD）活性及基因表达的影响。结果发现,M－CSF可以减轻tbOOH对RAW264．7细胞的氧化损伤;M－CSF可以提高细胞总SOD活性;M－CSF还可增强RAW264．7细胞MnSOD mRNA的表达,且这一诱导作用可被放线菌素D（actinomycinD)阻断。提示M－CSF可通过诱导细胞MnSOD表达而减轻RAW264．7细胞的氧化损伤。     

人参总皂甙对小鼠造血细胞增殖的影响

应用ＭＴＴ比色分析法证实,人参总皂甙在一定浓度内能促进小鼠骨髓造血细胞的增殖。应用流式细胞术的研究提示,人参总皂甙能促进骨髓造血细胞进入细胞增殖周期。造血祖细胞体外培养的研究表明,人参总皂甙能促进小鼠多种造血祖细胞的集落形成。综述上研究结果提示,人参总皂甙可能通过促进髓造血细胞进入细胞增殖周期以及促进造血祖细胞增殖等途径。刺激血细胞的生成,进而发挥其临床“补气生血”作用。     

云芝多糖增强巨噬细胞M—CSF的表达与分泌

为揭示云芝多糖作用与巨噬细胞Ｍ－ＣＳＦ表达与分泌的关系,采用活性测定,斑点杂交等方法,将云芝多糖对小鼠腹腔巨噬细胞Ｍ－ＣＳＦ表达及分泌的影响进行了探讨。结果显示,腹腔注射云芝多糖可以提高小鼠腹腔巨噬细胞培养上清中的Ｍ－ＣＳＦ活性,并使巨噬细胞Ｍ－ＣＳＦ ｍＲＮＡ的含量增加;应用ｍＲＮＡ合成抑制剂及蛋白合成抑制剂的研究发现,ａｃｔｉｎｏｍｙｃｉｎ Ｄ及ｃｙｃｌｏｈｅｘｉｍｉｄｅｆ均能阻断云芝多糖对小     

登革1～4型病毒prM-E基因的双价和四价重组质粒DNA在小鼠中的免疫原性观察

目的 在鼠模型中观察登革（DEN）病毒双价和四价重组质粒DNA的免疫原性，为登革多价DNA疫苗的研究奠定基础。方法采用可去除内毒素的试剂盒大量提取质粒DNA。将双价质粒DNA及将它们配伍后再与鼠源GM-CSF进行联合免疫，免疫途径采用肌肉注射，并加以电刺激，以提高质粒DNA的摄入效率。于初次免疫后第2和4周各加强免疫1次。然后在小鼠中分别测定针对登革1-4型病毒的体液和细胞免疫应答水平。采用间接免疫荧光法和中和试验测定血清抗体效价，细胞免疫应答水平通过测定脾淋巴细胞的增殖指数和其分泌的IFN-T浓度进行评价。细胞浸润实验通过对免疫部位的肌肉进行HE染色确定。结果小鼠在初次免疫后第4周即开始产生针对DEN1～4型病毒的抗体，随着时间的延长，抗体效价逐渐上升。第14周中和抗体效价最高达1：32。淋巴细胞的刺激指数及其分泌IFN-T的浓度均显著高于对照组。GM-CSF对体液免疫应答有促进作用，但对细胞免疫应答无显著的促进作用。结论本研究所构建的双价和四价重组质粒DNA在鼠模型中具有较好的免疫原性，这为登革多价DNA疫苗的研究奠定了基础。     

集落刺激因子-1受体在小鼠皮层神经元的表达

对体外培养神经元是否具有表达，合成集攻刺激因子受体的能力进行观察。方法在用免疫双标记的方法确定培养细胞系神经元的基础上，采原位杂交组织化学和免疫组织化学的方法，在ｍＲＮＡ和蛋白两个水平检查神经元是否拥有集落刺激因子受体。     

小鼠异基因造血干细胞移植后使用G-CSF对aGVHD的影响

 目的: 探讨小鼠异基因造血干细胞移植(allo-HSCT)后使用粒细胞集落刺激因子(G-CSF)对急性移植物抗宿主病(aGVHD)的影响及其可能的机制。方法: 以雄性C57BL/6小鼠(H-2^b)为异基因供鼠,雄性BALB/c小鼠(H-2^d)为同基因供鼠;以接受8 Gy[⁶⁰Co] γ射线全身照射(TBI)预处理雌性BALB/c小鼠为受鼠,并随机分为7组:单纯TBI组、同基因骨髓+脾细胞移植(Syn-BMST)组、异基因骨髓移植(allo-BMT)组、异基因骨髓+脾细胞移植(allo-BMST)组、Syn-BMST后G-CSF给药(Syn-BMST+G-CSF)组、allo-BMT后G-CSF给药(allo-BMT+G-CSF)组和allo-BMST后G-CSF给药(allo-BMST+G-CSF)组,各G-CSF给药组从移植后第1天(+1 d)开始皮下注射G-CSF 2 μg/d,观察至+60 d。比较各组生存时间、aGVHD发生情况和病理改变,流式细胞术检测骨髓H2-Kb⁺细胞百分率(异基因嵌合率),比较allo-BMST和allo-BMST+G-CSF组+10 d时血清细胞因子(IL-2、IL-4、IFN-γ和TNF-α)水平、脾总有核细胞数(SpTNC)和脾细胞免疫表型的差异。结果: 单纯TBI组小鼠于照射后9~15 d死于造血衰竭,其余各组+10 d时均100%获得造血重建,随机抽取2只异基因骨髓移植受鼠,+30 d供者细胞嵌合率分别为99.8%和99.4%,表明清髓性allo-HSCT模型建立成功。Syn-BMST、Syn-BMST+G-CSF、allo-BMT和allo-BMT+G-CSF组小鼠观察至+60 d均未发生aGVHD。与allo-BMST组相比,allo-BMST+G-CSF组受鼠出现aGVHD时间早、程度重、病理改变严重、存活时间明显缩短(P<0.05)、+10 d SpTNC明显增加(P<0.05)、脾脏NK细胞显著扩增 (P<0.01)、DC1/DC2比值减低(P<0.05),而2组血清IL-2、IL-4、IFN-γ和TNF-α水平差异无统计学意义。结论: 移植后使用G-CSF对小鼠异基因单纯骨髓移植后aGVHD无明显影响,但能显著加重allo-BMST后aGVHD的严重程度并缩短受鼠生存时间,该效应可能与G-CSF诱导供鼠NK细胞扩增有关,提示临床allo-HSCT后早期使用G-CSF可能触发或加重aGVHD的风险。     

巨噬细胞集落刺激因子及其受体mRNA在人卵泡颗粒细胞和胎盘及子宫内膜中的表达

目的： 观察巨噬细胞集落刺激因子（MCSF）及其受体（MCSFR）在人卵泡颗粒细胞、胎盘及子宫内膜细胞中的表达情况，进一步了解它们在生殖生理中的作用。方法： 收集人卵泡颗粒细胞、胎盘及子宫内膜细胞标本。采用逆转录-聚合酶链式反应（RT-PCR）及原位逆转录-聚合酶链式反应（in situ RT-PCR）技术观察MCSF及MCSFR mRNA的表达情况。结果： 在人卵泡颗粒细胞、胎盘及子宫内膜细胞中，RT-PCR显示有MCSF及MCSFR的表达，原位RT-PCR也进一步证实了这一点,并定位于细胞浆。结论： MCSF可能参与了人卵泡颗粒细胞、胎盘及子宫内膜细胞生长、发育的调节，在生殖生理过程中可能起一定的作用。     

巨噬细胞集落刺激因子受体在白血病细胞系中的表达和作用

目的 探讨巨细胞集落刺激因子受体（M－CSF－R）在人白血病细胞系中的表达及其作用。方法 采用ABC免疫酶标技术,间接免疫荧光染色,流式细胞计和蛋白免疫印迹研究了4株人白血病细胞系（J6-1,J6-2)来源于人粒－单型白血病,HL－60为来源于人急性粒细胞白血病的髓样细胞系,K562为来源于人慢性白血急变期胸水的髓样细胞系）和正常人外周单个核细胞及正常人脐带血单个核细胞M－CSF－R的分布,表达,分子大小及其作用。结果 正常人外周血单个核细胞未见M－CSF－R的表达,经PHA刺激后有低水平的表达;4株人白血病细胞系都高表达M－CSF－R,分布于细胞膜,细胞质和细胞核,J6－1,J6－2,K562和HL－60细胞质和胞核M－CSF－R（M－CSF－cnR)平均阳性率分别为52．3％,44．3％,28．0％,65．3％;胞膜M－CSF－R（M－CSF－mR)阳性率分别为78．9％,72．0％,54．9％,58．0％;高于正常人外周血单个核细胞的表达水平。HL－60细胞质和胞核中有一种相对分子质量为120000的M－CSF－R分子;4种白血病细胞表达的M－CSF－cnR的半衰期分别高于正常人脐带血单个核细胞经PHA诱导表达的M－CSF－cnR的半衰期;J6－1,J6－2,HL－60细胞表达的M－CSF－mR的半衰期亦明显延长。抗M－CSF－R单抗和人重组的M－CSF－R可溶性受体均能使4种白血病细胞的增殖阻断在G0／G1期,并能抑制其在软琼脂上形成集落的能力。结论 M－CSF－R在人白血病细胞系的表达呈异质性;胞内M－CSF－R的蓄积可能是白血病细胞M－CSF－R降解速率下降的结果,也可能是白血病细胞增殖的内源信号;M－CSF－R介导的信号是HL－60细胞增殖的重要和必要信号。     

肝癌细胞增殖受M-CSF胞内和胞外自分泌的双重调控

目的：研究胞内M-CSF及其受体在肝癌SMMC 7721细胞的表达与性质,探讨胞内M-CSF对SMMC 7721细胞增殖的影响及其机制。 方法： 以高表达M-CSF的人肝癌细胞系（SMMC 7721细胞）为模型，以免疫组化、流式细胞计数、反义技术与蛋白印迹等方法观测胞内M-CSF对SMMC 7721细胞增殖的影响及其机制。 结果： M-CSF 及其受体主要在SMMC 7721细胞的胞质、胞核中表达，胞内的M-CSF的相对分子量为20 000，M-CSFR的相对分子量为120 000；免疫共沉淀分析证明M-CSF在细胞内与M-CSFR以复合物的形式存在；M-CSF的单克隆抗体及其反义寡聚核苷酸能抑制SMMC 7721细胞的增殖、下调cyclinD1/E的表达和上调p16的表达，且M-CSF的单克隆抗体及其反义寡聚核苷酸的联合使用能进一步加强对SMMC 7721细胞抑制作用和增加下调cyclinD1/E和上调p16的表达幅度。 结论： SMMC 7721细胞受M-CSF胞外自分泌和胞内自分泌的双重调控。     

Preparation of quiescent human monocytes for studies of colony-stimulating factor responsiveness

Summary A method is described for isolating by adherence human peripheral blood monocytes and rendering them quiescent. Quiescence is achieved by depriving the cells of human serum and hematopoietic growth factors. These quiescent monocytes express very low levels of immediate early response gene mRNAs and are thus useful in studies of signal transduction with colony stimulating factors.     

Differential augmentation of in vivo natural killer cytotoxicity in normal primates with recombinant human interleukin-1 and granulocyte-macrophage colony-stimulating factor.

The effect of recombinant human interleukin-1 (IL-1) alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and combined factor therapy (CFT) on Rhesus monkey peripheral blood natural killer (NK) activity in vivo was compared. During a 14-day treatment period, IL-1-treated animals demonstrated a 170% increase in NK activity against K562 target cells by day 4, reaching maximal levels (300%) at day 16, and returning to baseline by day 30. NK activity of GM-CSF-treated monkeys increased slightly (60-100%) during days 4-12, as did saline-treated monkeys, but returned to baseline values by day 16. A delayed increase in NK activity resulted after GM-CSF treatment, reaching a peak (260%) on day 23 and remaining elevated through day 39. CFT resulted in a bimodal response pattern, with two peaks of NK activity: one at day 16 and a second at day 39. The first peak of activity (223%) was significantly less than the activity attained with IL-1 alone; the second peak (300%) was of greater duration and occurred later than the peak observed in GM-CSF-treated monkeys. Unlike IL-1, GM-CSF treatment did not lead to a immediate stimulation of NK activity; augmentation was delayed by more than 7 days post treatment. CFT results suggest that GM-CSF reduced the direct NK response to IL-1; while IL-1 led to an enhanced delayed NK response. Therefore, IL-1 and GM-CSF augment NK activity through different but interrelated pathways.     

粒细胞集落刺激因子调节T淋巴细胞免疫功能的实验研究

目的研究粒细胞集落刺激因子(G-CSF)对T淋巴细胞免疫功能的调节作用。方法以15例健康供者为对象,在其应用G-CSF前后,用流式细胞仪(FCM)检测外周血T淋巴细胞表面免疫标记、Annexin V及细胞内细胞因子的表达,用淋巴细胞转化试验检测T淋巴细胞的增殖反应。结果供者用G-CSF后,CIM^ 、CD8^ 细胞数无明显变化,CIM^ 、CD8^ 细胞内IFN-γ表达降低,IL-4表达增高;T细胞对丝裂原的增殖反应降低。Th1和Th2细胞表面Annexin V的表达无显著性差异。结论G-CSF制了丝裂原刺激T细胞的功能,使供者T细胞向Th2型细胞转化,细胞凋亡不是Th1／Th2转化的机制。     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not granulocyte colony-stimulating factor (G-CSF) induces plasma membrane expression of proteinase 3 (PR3) on neutrophils in vitro

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener's granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-alpha)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-alpha. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism.     

Effects of macrophage colony-stimulating factor (M-CSF) on anti-fungal activity of mononuclear phagocytes against Trichosporon asahii

Trichosporon asahii is an emerging opportunistic pathogen in immunocompromised patients. Little is known about the mechanisms of host defence against T. asahii. We investigated the fungicidal activity of human peripheral blood monocytes and murine peritoneal macrophages against T. asahii isolates, and the effects of M-CSF on the anti-fungal activity of mononuclear phagocytes. We also established a neutropenic mouse model of disseminated trichosporonosis with T. asahii. M-CSF enhanced the phagocytic fungicidal activity of mononuclear cells, and infected mice treated with human M-CSF at 10 x 106 U/kg showed a significant improvement in survival rate, with fewer fungal colony counts in the lung compared with control mice. Mice treated with human M-CSF showed higher concentrations of tumour necrosis factor-alpha (TNF-alpha) in the lung and plasma compared with control mice. The survival rate was significantly reduced in mice treated with anti-mouse TNF-alpha. Our results showed that M-CSF enhanced the fungicidal activity of mononuclear phagocytes partly by production of TNF-alpha, and suggest that the administration of M-CSF to patients with disseminated trichosporonosis may be a useful adjunct to conventional anti-microbial therapy and prophylaxis.     

Distribution of the hematopoietic growth factor G‐CSF and its receptor in the adult human brain with specific reference to Alzheimer's disease

The granulocyte colony‐stimulating factor (G‐CSF), being a member of the hematopoietic growth factor family, is also critically involved in controlling proliferation and differentiation of neural stem cells. Treatment with G‐CSF has been shown to result in substantial neuroprotective and neuroregenerative effects in various experimental models of acute and chronic diseases of the central nervous system. Although G‐CSF has been tested in a clinical study for treatment of acute ischemic stroke, there is only fragmentary data on the distribution of this cytokine and its receptor in the human brain. Therefore, the present study was focused on the immunohistochemical analysis of the protein expression of G‐CSF and its receptor (G‐CSF R) in the adult human brain. Since G‐CSF has been shown not only to exert neuroprotective effects in animal models of Alzheimer's disease (AD) but also to be a candidate for clinical treatment, we have also placed an emphasis on the regulation of these molecules in this neurodegenerative disease. One major finding is that both G‐CSF and G‐CSF R were ubiquitously but not uniformly expressed in neurons throughout the CNS. Protein expression of G‐CSF and G‐CSF R was not restricted to neurons but was also detectable in astrocytes, ependymal cells, and choroid plexus cells. However, the distribution of G‐CSF and G‐CSF R did not substantially differ between AD brains and control, even in the hippocampus, where early neurodegenerative changes typically occur.     

Ultrastructural effects of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) on rat neutrophils and monocytes

Human granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF) were administered intravenously to rats, and their effects on neutrophils and monocytes were examined by electron microscopy. G-CSF increased the number of cytoplasmic granules in neutrophils. It also enhanced maturation of the nuclear shape in the neutrophils, while chromatin condensation and peroxidase distribution remained immature. M-CSF induced proliferation of monocytes in peripheral blood and bone marrow, but did not affect morphology or distribution of peroxidase reactivity. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.