粒细胞集落刺激因子减轻混合骨髓移植后移植物抗宿主病的实验研究

Objective: How to reduce the incidence and severity of acute graft-versus-host disease (aGVHD) is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (allo-BMT). The low incidence of severe aGVHD observed in allogeneic peripheral blood stem cell transplantation (allo-PBSCT), which may be related to modulating immune function of T lymphocytes by granulocyte colony-stimulating factor (G-CSF) primed donors. The study aimed to explore whether aGVHD could be alleviated by syngeneic bone marrow mixed with G-CSF-mobilized H-2 haploidentical marrow grafting. Methods: Female BALB/c mice and neonatal BALB/c mice were recipients and male (BALB/c × C57BL/6)F1(BCF1) mice were donor mice respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g body weight or saline for 6 days, and splenocytes were harvested on day 6. Spleen index (SI) represented GVHD in neonatal mice after the intraperitoneal injection of mixed spleen cells. Lethally irradiated (60Co, 8.5 Gy) adult mice were transplanted with a mixture of syngeneic plus G-CSF-mobilized (control diluents) H-2 haploidentical marrow cells. Survival time and survival rate of the recipients were observed after mixed marrow transplantation (MBMT). GVHD was assessed by observing signs of weight loss, ruffled fur, diarrhea and histological change of skin, liver and small intestines. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (IL-2, IL-4 and INF-γ). Fluorescence-activated cell sorting (FACS) analysis was used to detect T cells phenotype. Results: (1) The neonatal mice subject to injection of 2:1 and 1:1 mixed spleen cells and H-2 haploidentical spleen cells all suffered from aGVHD. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes was dramatically reduced. (2) The aGVHD signs and histological change were observed in most mice of 2:1 and 1:1 MBMT groups. However, the survival time of G-CSF-mobilized MBMT was longer than in control groups and these mice had signs of moderate GVHD. (3) L3T4 cells and relative ratio in both subsets was significantly reduced in G-CSF-treated donor mice. The total number of Thy1.2 and lyt2 cells was increased after G-CSF pretreatment of donors, but no statistical difference. (4) The supernatants from a primary MLR were collected at 48 h for cytokine measurement. The results showed an increased production of IL-4 and a decreased production of IL-2 and INF-γ after stimulating with concanavalin A for 48 h. Conclusion: The GVHD could be reduced using syngeneic bone marrow mixed with H-2 haploidentical marrow cells. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes or marrow cells could be further moderated, which is associated with increased IL-4 production and decreased IL-2 and INF-γ production.     

重组人粒细胞集落刺激因子致急性肾衰竭

1名54岁男性失代偿期肝硬化患者,皮下注射重组人粒细胞集落刺激因子(rhG-CSF)200μg,1次/d。用药后第2天患者出现尿色变深,第4天出现眼睑水肿,肉眼血尿、少尿等症状。BUN由4.8mmol/L升至7.9mmol/L(最高13.9mmol/L),Cr由113μmol/L升至154μmol/L(最高308μmol/L)。停用rhG-CSF,给予还原型谷胱甘肽、硫普罗宁、呋塞米等对症支持治疗。2周后肾功能恢复正常。     

重组人粒细胞集落刺激因子对健康供者免疫细胞的影响及其应用安全性

目的:观察重组人粒细胞集落刺激因子(rhG-CSF)在动员造血干细胞时对健康供者外周血T细胞表面活化信号(CD4 CD28 ,CD8 CD28 )、活化T细胞(CD4 CD25low)和调节性T细胞(CD4 CD25high)的影响及其应用的安全性。方法:30例造血干细胞健康供者[男19,女11,中位年龄28(14～56)岁]皮下注射rhG-CSF5μg/(kg.d),连续4～6d。用药前及停药后第1天、第3天及第7天用流式细胞术检测供者外周血CD4 T细胞和CD8 T细胞表面CD28表达的相对荧光强度(RFI),以及CD4 CD25low和CD4 CD25highT细胞分别在CD4 T细胞中的百分比;并在用药前及停药后第1天、第7天及第30天进行外周血白细胞、血小板、肝功能、肾功能以及脾超声检查。结果:CD4 CD28T细胞和CD8 CD28T细胞相对荧光强度表达在用药前分别为14.91±6.10和11.10±3.74,停药后第3天分别降至11.93±5.39和8.53±3.74;差异有统计学意义(P=0.034,P=0.033)。CD4 CD25lowT细胞在CD4 T细胞中百分比,用药前为(21.4±8.87)%,停药后第3天降至(18.23±5.89)%;差异无统计学意义(P>0.05)。CD4 CD25highT细胞在CD4 T细胞中百分比,用药前为(4.16±1.62)%,停药后第1天升至(6.43±2.46)%,差异有统计学意义(P=0.000)。白细胞计数和脾超声检查用药前分别为(5.91±1.02)×109/L和(33.76±2.76)cm2,停药后第1天分别升至(34.13±8.07)×109/L和(46.85±4.53)cm2;血小板计数用药前为(228.07±73.69)×109/L,停药后第7天降至(158.27±40.69)×109/L,差异均有统计学意义(P均=0.000)。ALT和Cr用药前分别为(28.23±7.69)IU/L和(60.70±15.86)μmol/L,停药后第1天分别为(27.17±7.23)IU/L和(61.10±16.38)μmol/L,差异均无统计学意义(P均>0.05)。结论:rhG-CSF能减弱CD28的表达,降低CD4 CD25low和增加CD4 CD25high在CD4 T细胞中百分比,停药后第7天恢复至动员前水平,对健康供者肝肾功能无不利影响。     

射干麻黄汤对实验性哮喘豚鼠嗜酸性粒细胞凋亡的影响

目的:探讨射干麻黄汤对嗜酸性粒细胞凋亡的影响。方法:以卵白蛋白致敏法制作豚鼠哮喘模型并用射干麻黄汤进行干预,实验结束后分析各组动物的嗜酸性粒细胞凋亡情况。结果:实验组豚鼠嗜酸性粒细胞显著下降、凋亡明显增加与模型组比较有统计意义(P<0.01或P<0.05)。结论:射干麻黄汤可有效促进嗜酸性粒细胞凋亡,从而达到治疗哮喘的目的。     

rhG-CSF拮抗糖尿病脑缺血后神经细胞凋亡的研究

目的验证重组人粒细胞集落刺激因子(rhG-CSF)是否能改善局灶性脑缺血糖尿病大鼠的神经功能,并观察对神经细胞凋亡的影响。方法线栓法复制糖尿病大鼠大脑中动脉栓塞模型,rhG-CSF干预组连续皮下注射rhG-CSF,50μg·kg-1·d-1,进行神经功能缺损评分,采用TUNEL染色计数脑缺血周边区神经细胞凋亡数。结果rhG-CSF干预组神经功能缺损评分明显低于对照组(P<0.01);rhG-CSF干预组缺血周边区的TUNEL阳性细胞数均明显少于对照组(P<0.01)。结论rhG-CSF可拮抗糖尿病脑缺血之后神经细胞凋亡,改善神经功能。     

预激方案治疗难治急性髓系白血病的临床观察

目的探讨预激方案治疗难治急性髓系白血病的临床疗效和毒副作用。方法选择2004年7月-2005年11月在该科治疗的难治急性髓系白血病17例,2例为骨髓增生减低者,1例为71岁患者伴冠心病,用预激方案前未经化疗。其余14例经过传统的MA、HA、DA等方案治疗1疗程,未取得完全缓解,造血恢复后仍体质差,全血细胞减少。预激方案ACR10-14mg/m^2,Qd,第1-8天,Ara-c 10mg/m^2,Q12h,第1-14天,G-CSF200μg/m^2,Qd,第1-14天。结果17例经上述治疗达CR11例（64.7%）,PR3例（17.6%）,NR3例（17.7%）。结论预激方案治疗急性髓系白血病完全缓解率与传统的的HA、DA方案相似,但不良反应小、使用安全,为老年、低增生性及体质差的急性髓系白血病患者提供了新的治疗方案。     

G-CSF in the Biology and Treatment of Acute Myeloid Leukemias

Granulocyte colony-stimulating factor (G-CSF) is an hemopoietic growth factor produced by fibroblasts, monocytes and endothelial cells. The role of G-CSF in the biology of acute myeloid leukemia (AML) has been investigated by several authors, who have demonstrated receptor mediated enhanced proliferation of AML blasts in vitro, in the presence of G-CSF. This effect is further increased by addition of other cytokines such as GM-CSF, IL3, IL4, Stem cell factor (SCF), while Tumor Necrosis Factor (TNF) and Transforming Growth Factor β1 (TGF β1) seem to exert an inhibitory activity. An autocrine production of G-CSF by AML cells, a paracrine production by accessory cells and a protective effect displayed by G-CSF against programmed cell death could partially contribute to explain the pathogenesis of AML. In vivo, G-CSF has been used after chemotherapy in AML, in order to improve hemopoietic recovery in patients at high risk of infection. Current studies are focusing on better definition of the role of G-CSF, as such or combined with other biological modifiers, in dose intensification and autologous bone marrow or peripheral blood stem cell transplantation.     

联合应用成纤维细胞介导的G-CSF基因疗法及IL-2基因疗法对肿瘤的治疗作用

本实验观察了成纤维细胞介导的G-CSF基因疗法对大剂量化疗后造血功能损伤小鼠的恢复作用。结果发现:接受G-CSF基因治疗的实验小鼠外周血白细胞尤其是中性粒细胞降低程度明显减弱,恢复速度明显加快;并可明显促进血小板的恢复,但作用较缓;其脾脏和骨髓CFU-GM、CFU-MK、CFU-S水平显著地高于对照组,表明成纤维细胞介导的G-CSF基因疗法可显著降低大剂量化疗后造血损伤程度,并明显加速受损的造血功能的恢复。     

Generalised glucosephosphate isomerase (GPI) deficiency causing haemolytic anaemia,neuromuscular symptoms and impairment of granulocytic function: a new syndrome due to a new stable GPI variant with diminished specific activity (GPI Homburg)

A new glucosephosphate isomerase (GPI) variant is described which is characterised by very low specific activity in erythrocytes, granulocytes and muscle tissue, nearly normal stability, normal kinetic properties and a decreased electrophoretic mobility. The propositus suffers from a complex syndrome involving erythrocytes (congenital haemolytic anaemia), granulocytes (decreased production of superoxide anion and reduced bactericidal activity in vitro) and the neuromuscular system (myopathy, mental retardation). It is suggested that the clinical syndrome results from generalised GPI deficiency due to a decreased specific activity of the variant enzyme, which cannot be compensated by an increase of de-novo synthesis of GPI protein even in cells exhibiting active protein synthesis such as granulocytes and muscle cells.Abbreviation GPI glucosephosphate isomerase     

左归丸对环磷酰胺诱导小鼠白细胞降低的保护作用及其机制

目的探讨左归丸对环磷酰胺诱导小鼠白细胞降低的保护作用及其可能机制。方法♂昆明种小白鼠120只,随机分为5组,每组24只。空白对照组和模型组给予NS,治疗组分别给予左归丸1.8、3.65、.4 g.kg-1(Ⅰ~Ⅲ组),ig,连续10 d;于d 4~6空白对照组给予NS,模型组和Ⅰ~Ⅲ组给予环磷酰胺50 mg.kg-1,ip,qd。各组小鼠中的12只于给药前1 d及d 71、1、15尾静脉取血计数WBC,其余12只小鼠于d 11检测骨髓有核细胞计数(BMC)、脾脏指数和胸腺指数、脾集落形成单位(CFU-S)、血清粒细胞集落刺激因子(G-CSF)和白细胞介素6(IL-6)活性。结果试验d 7时各组小鼠的WBC均明显低于空白对照组,在d 11、15时,治疗的WBC最高比模型组分别提高93%和40%(P<0.01)。d 11时,治疗组的BMC、脾脏指数、CFU-S、G-CSF最高比模型组分别提高92%、83%、85%、45%和45%(P<0.01)。结论左归丸对环磷酰胺所致的白细胞降低具有保护作用,该作用可能与升高血清G-CSF活性有关。