转粒细胞-巨噬细胞集落刺激因子基因真核表达质粒的构建及生物活性鉴定

目的　构建小鼠转粒细胞 巨噬细胞集落刺激因子 (mGM CSF)真核表达质粒 pcDNA3 GM CSF ,转染红白血病细胞系FBL 3,并鉴定其活性。方法　采用分子克隆技术 ,构建mGM CSF真核表达质粒 pcDNA3 GM CSF ,电穿孔法将其导入红白血病细胞系FBL 3,G4 18筛选G4 18抗性细胞 ,限制性稀释法筛选G4 18抗性细胞克隆。PCR和RT PCR方法鉴定GM CSF基因整合和稳定表达。骨髓造血祖细胞增殖实验和骨髓造血祖细胞集落刺激实验鉴定其生物学活性。结果　采用PCR方法扩增mGM CSFcDNA序列 ,BamHⅠ和EcoRⅠ双酶切后装入 pcDNA3质粒 ,构建真核表达质粒 pcDNA3 GM CSF ,酶切和测序结果与预期相符 ,无插入、丢失、突变 ,方向正确。PCR和RT PCR结果显示 ,GM CSF基因整合到受体细胞染色体中并稳定表达。表达GM CSF的FBL 3 GM CSF细胞培养上清可明显刺激小鼠骨髓单个核细胞增殖 ,并能刺激干细胞形成克隆 ,形成克隆数为 (5 4 .6 7± 4 .83)个 ,形成率为 0 .5 4 7%。结论　构建mGM CSF真核表达质粒pcDNA3 GM CSF ,获得稳定表达该基因并具有生物学活性的细胞克隆 ,为制备转GM CSF基因瘤苗 ,探索白血病免疫治疗的可行性奠定基础     

粒细胞巨噬细胞集落刺激因子与感染

粒细胞巨噬细胞集落刺激因子(GMCSF)是造血细胞因子家族中的成员,是一种能在造血干细胞、祖细胞和成熟细胞不同水平上促其生成的多向性造血因子和内源性调节因子。GMCSF作为一种内源性调节因子在抗感染免疫中对某些细菌、真菌、病毒和寄生虫感染中有重要作用。具有潜在的临床应用价值     

粒细胞-巨噬细胞集落刺激因子的研究进展

粒细胞一巨噬细胞集落刺激因子（GM—CSF）主要由体内激活的T细胞、单核巨噬细胞等产生。近年研究发现它是一种有广泛免疫活性的效应因子,在激活免疫反应中起重要作用,参与特异性抗体的产生。GM—CSF主要促进粒、巨噬细胞增殖、分化及功能成熟,提高宿主的防御能力,而且可以活化巨噬细胞产生依赖性细胞介导的细胞毒效应（ADCC）和多形性中性粒细胞（PMN）产生补体介导的吞噬作用,以及增强PMN的各种功能。GM—CSF还能提高C3bi及Fcr受体的表达,现就其近年来的研究进展作一综述。     

粒细胞、粒巨噬细胞集落刺激因子在新生儿期的应用

新生儿感染的发病率及病死率较高，吞噬细胞系统不成熟是主要原因这一，粒细胞，粒巨噬细胞集落刺激因子可刺激粒子巨噬细胞增殖，分化、增强中性粒细胞和单核巨噬细胞功能，临床上应用重组人类集落刺激因子治疗和预防新生儿感染取得了令人鼓舞的效果。     

粒细胞、粒巨噬细胞集落刺激因子在新生儿期的应用

新生儿感染的发病率及病死率较高，吞噬细胞系统不成熟是主要原因之一。粒细胞、粒巨噬细胞集落刺激因子可刺激粒巨噬细胞增殖、分化，增强中性粒细胞和单核巨噬细胞功能。临床上应用重组人类集落刺激因子治疗和预防新生儿感染取得了令人鼓舞的效果。     

粒细胞-巨噬细胞集落刺激因子联合乙肝疫苗对宫内感染HBV携带儿童治疗机制初探

目的从细胞因子水平初步探讨粒细胞一巨噬细胞集落刺激因子(GM—CSF)联合乙肝疫苗对宫内HBV感染携带儿童的疗效机制。方法采用ELISA和半定量逆转录-聚合酶链反应(RT—PCR)，对12例GM—CSF联合乙肝疫苗治疗后儿童、6例乙肝免疫球蛋白(HBIG)联合乙肝疫苗治疗后儿童及9例未治疗的宫内感染儿童外周血单个核细胞在PHA LPS、HBsAg及无刺激物时γ-干扰素、白细胞介素(IL)-4分泌及mRNA表达水平进行检测。结果与对照组比较，GM-CSF联合乙肝疫苗治疗儿童γ-干扰素自发分泌显著增加(P=0．017)，HBIG联合乙肝疫苗治疗儿童IL-4 mRNA转录显著降低(P=0．002)。结论GM—CSF联合乙肝疫苗治疗宫内感染HBV慢性携带儿童时HBV受抑制可能与γ-干扰素产生增多有关，HBIG联合乙肝疫苗治疗时IL-4转录受抑不影响HBV复制，提示慢性HBV感染治疗应以增强Th1应答为主。     

重组人粒-巨噬细胞集落刺激因子治疗儿童药物性粒细胞缺乏症18例

重组人粒 巨噬细胞集落刺激因子 (ｒＨｕＧＭ ＧＳＦ)能特异性刺激粒细胞和巨噬细胞的分化、增殖和成熟 ,并能提高效应细胞的免疫活性 ,起到加强机体免疫力和对抗感染的作用。骨髓抑制是抗癌药物的主要副作用 ,是化学治疗 (简称化疗 )中最显著的障碍因素 ,在临床上表现为粒细胞减少以及与之相关的感染。目前临床已将ｒＨｕＧＭ ＣＳＦ广泛用于强烈化疗及骨髓移植所致的白细胞减少症或粒细胞缺乏症。我们于 2 0 0 0年 3～ 6月将厦门特宝生物工程有限公司研制的ｒＨｕＧＭ ＣＳＦ(商品名 :特尔立 )用于化疗后药物性粒细胞减少症的治疗。对象和…     

心肌疾病抗ADP/ATP载体抗体与粒细胞-巨噬细胞集落刺激因子的临床研究

目的探讨病毒性心肌炎（ＶＭＣ）和扩张型心肌病（ＤＣＭ）的自身免疫发病机制。方法采用免疫印转和放射免疫技术检测了３０例ＶＭＣ、１４例ＤＣＭ患儿血浆中抗心肌线粒体ＡＤＰ／ＡＴＰ运载蛋白（ＡＮＴ）抗体和粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）。结果ＶＭＣ和ＤＣＭ患儿抗ＡＮＴ抗体和ＧＭ－ＣＳＦ的阳性率分别为７３％和５７％，而２５例正常健康儿分别为０和１２％（Ｐ均＜０．０１），且抗ＡＮＴ抗体与ＧＭ－ＣＳＦ的血浆水平呈正相关（ｒ＝０．４０９１，Ｐ＜０．０５）。结论小儿ＶＭＣ和ＤＣＭ的发病与心肌的自身免疫损伤有关，抗ＡＮＴ抗体和ＧＭ－ＣＳＦ均参与了这一过程。抗ＡＮＴ抗体可作为小儿ＶＭＣ和ＤＣＭ的一种特异性诊断指标。     

新生儿肺炎血清粒细胞集落刺激因子的研究

粒细胞集落刺激因子（G－CSF）是一种体内产生的糖蛋白生长因子。在人体被细胞感染后明显升高。我们观察36例肺炎新生儿血清G－CSF水平。观察结果表明新生儿肺炎血清G－CSF升高，与对照组比较有非常显著差异（P＜0．001）。G－CSF阳性表明体内有细菌感染存在。临床上我们根据G－CSF水平酌情合理使用抗生素，并取得较好效果，因此我们认为血清G-GSF的检测对新生儿感染性疾病的抗生素应用有指导意义。     

白血病患儿重组粒细胞集落刺激因子血药浓度监测及临床意义

目的研究急性白血病患儿反复应用重组粒细胞集落刺激因子(rhG-CsF)的药代动力学及其与临床的关系。方法应用酶联免疫吸附测定(ELIsA)方法检测46例白血病患儿的血清G-CSF浓度。结果获得29例患儿用rgG-CSF后的标本,其血清G-CSF浓度为(5.335±5.597)μg/L。反复应用rhG-CSF的患儿在下次用药前血清G-CSF水平恢复正常;少数未应用rgG-CSF患儿可检测到较高浓度的G-CsF。血清G-CSF浓度与粒细胞绝对计数值呈负相关(r=-0.8746,P＜0.01)。结论rhG-CSF的药代动力学中存在有粒细胞介导的清除机制,反复应用无药物蓄积作用。     

Granulocyte-macrophage colony stimulating factor does not improve neutrophil oxidative metabolism in a patient with variant X-linked chronic granulomatous disease

Variant X-linked chronic granulomatous disease (CGD) is characterised by a decreased but still measurable respiratory burst and cytochrome b content of phagocytes resulting in a clinically milder form of the disease. We examined the in vivo effect of recombinant human granulocyte-macrophage colony stimulating factor (rh-GM-CSF) on the neutrophil functions of a patient treated for liver abscess. The number of white blood cells was markedly increased at the highest dose of GM-CSF injected (30 g/kg per day). This was mainly due to a large increase in eosinophils and to a lesser extent in neutrophils. No change in the deficient neutrophil respiratory burst nitroblue tetrazolium (NBT)-reduction, superoxide (O ₂ ^– )-production and cytochrome b content was observed during 6 weeks of therapy with increasing doses of GM-CSF. No significant clinical improvement of the liver abscess was observed during treatment with GM-CSF.     

Use of recombinant human granulocytemacrophage colony stimulating factor in an infant with reticular dysgenesis

We present the case of a 2-month-old infant with reticular dysgenesis who was treated with recombinant granulocyte-macrophage colony stimulating factor with the aim of stimulating granulopoiesis while awaiting bone marrow transplant.     

Protective effects of recombinant human granulocyte colony stimulating factor in a rat model of necrotizing enterocolitis

The role of cytokines and growth factors in the pathophysiology of neonatal necrotizing enterocolitis (NEC) is not defined clearly yet. The aim of this study was to determine the effects of recombinant human granulocyte colony stimulating factor (G-CSF) on intestinal cells in hypoxia-induced experimental NEC in rats. The study was experimented on Sprague Dawley rat pups. Group 1 (untreated, n = 7) rats were subjected to hypoxia–reoxygenation (H/O) and then were returned to standard conditions. Group 2 (G-CSF treated, n = 7) rats were subjected to H/O, and then were treated with G-CSF (100 μg/kg enterally) for 5 days. Group 3 was served as nonhypoxic controls. All animals were killed on day five, and histological examination was performed on intestinal samples. There were no histopathological changes in the control group. The histological findings in untreated rats were similar to those seen in neonatal NEC, with destruction of villi and crypts with extension to the muscularis layer. Intestinal damage was mild in group 2 and these histological changes were better than group 1, and worse than group 3. The mean of histologic grade of group 1 was 2.4 (range 2–3), and in the group 2, it was 1.2 (range 0–2). A difference was found when two groups were compared with each other (P < 0.05). In an experimental model of NEC, G-CSF could have a protective effect on intestinal damage.     

Isolated cutaneous response to granulocyte-monocyte colony stimulating factor in fatal idiopathic disseminated Bacillus-Calmette-Guerin infection

Severe disseminated Bacillus-Calmette-Guerin (BCG) infection is very rare and has been regarded as idiopathic when no immunodeficiency is present. This entity seems to be due to several new types of inherited abnormalities in the pathways important in defence against Mycobacteria. Although improvement with interferon-γ (IFN-γ) has been reported in some patients, to our knowledge there are no reports on the effect of other cytokines in the treatment of these patients. We report here the clinical response to IFN-γ and granulocyte-monocyte colony stimulating factor (GM-CSF) treatment in a patient with idiopathic disseminated BCG infection who failed to respond to multiple antimycobacterial agents. The patient showed partial and transitory response to IFN-γ, however, GM-CSF treatment led to rapid improvement of skin lesions within 2 weeks without any effect on the progression of the disease in the other organ systems. Conclusion The response of idiopathic disseminated Bacillus-Calmette-Guerin infection to granulocyte-monocyte colony stimulating factor treatment was limited to cutaneous lesions. Granulocyte-monocyte colony stimulating factor may have acted to promote wound healing or the levels of this factor achieved in other affected organs may have been inadequate. Received: 26 January 1999 / Accepted: 1 July 1999     

Stage-specific changes in the levels of granulocyte-macrophage colony-stimulating factor and its receptor in the biological fluid and organ of mouse fetuses

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that has neurotrophic and neuroprotective functions. However, its function in the mid- to late-gestational fetus remains unclear. We used enzyme-linked immunosorbent assay to analyze GM-CSF levels in cerebrospinal fluid (CSF), serum, and amniotic fluid of mouse fetuses. We also examined GM-CSF and receptor α (GM-CSFRα) levels in the fetal brain, liver, and placenta. GM-CSF peaked between embryonic day (E) 14 and E15 in the CSF. GM-CSF level was higher in the fetal serum than in the dam serum on E13 and decreased thereafter. GM-CSF and GM-CSFRα levels peaked between E13 and E15 in the brain. These results suggest that GM-CSF plays stage- and organ-specific roles in fetal development.     

Serum concentration of granulocyte colony stimulating factor in term and preterm infants

We measured serum granulocyte colony stimulating factor (GCSF) concentration and absolute neutrophil count in four groups of infants: (1) 15 healthy term newborn infants; (2) 21 healthy preterm newborn infants, with mean (SD) birth weight 1583 (533) g, and gestational age 32.0 (3.8) weeks; (3) 5 infected newborn infants; (4) 22 6-month-old control infants. Median (range) serum GCSF concentration was 132.2 (41.5–176.0) pg/ml in term infants, 51.5 (1.8–175.7) pg/ml in preterm infants and 138.9 (54.1–449.8) pg/ml in 6-month-old control infants, with a significant reduction in preterm infants, as compared to term and control infants. GCSF levels were significantly higher in the infected infants, as compared to healthy neonates. Conclusion A significant positive relationship was found in term and preterm infants between serum GCSF concentration and gestational age or birth weight. No relationship was found between serum GCSF concen tration and neutrophil count. The low GCSF baseline levels may contribute to the increased incidence and severity of infection in preterm infants. Received: 17 May 1996 and in revised form: 20 July 1996 / Accepted: 29 July 1996     

Safety and feasibility of granulocyte colony‐stimulating factor (G‐CSF) primed bone marrow (BM) using three days of G‐CSF priming as stem cell source for pediatric allogeneic BM transplantation

There are limited data on the optimal dosing and schedule of G‐CSF priming prior to BM harvest. We evaluated the safety and efficacy of three days of G‐CSF of primed BM from related pediatric donors. Forty‐five children were treated. All donors received 5 μg/kg per day of G‐CSF as a single subcutaneous injection for three consecutive days prior to the BM harvest. The median age of the donors was seven yr (range, 0.8–18) and no donor experienced major adverse events related to G‐CSF administration. The median age for the recipients was five yr (0.3–16 yr). Thirty‐five patients had non‐malignant disorders. The median dose of nucleated (TNC) and CD34+, CD3 cells infused per recipient weight was 5.4 × 10⁸/kg (range, 0.61–17), 4.7 × 10⁶/kg (range, 1.6–19), and 43.8 × 10⁶/kg (range, 1.8–95), respectively. All patients achieved neutrophil and platelets engraftment, at a median of 15 (range, 10–22) and 23 days (range, 13–111), respectively. At a median follow up of 60 months (range 12–100), the estimated five yr overall and EFS was 91% and 80%, respectively. Collection of BM following three days of G‐CSF priming from pediatric donors is safe and results in high TNC and CD34+ cell yield.