启动子Ptac与PsbA在鱼腥藻7120中表达hG-CSF的效率比较

为了比较外源性启动子Ptac与内源性启动子PsbA在鱼腥藻7120中表达外源基因时的效率,构建了分别含Ptac和PsbA两种启动子的穿梭表达载体pRL-PsbA-GCSF、pRL-Tac-GCSF;利用三亲结合转移法转化鱼腥藻7120,利用抗生素筛选,通过质粒提取和PCR方法鉴定,获得了分别由2种启动子驱动表达hG-CSF的转基因蓝藻,转基因藻中目的基因以质粒形式存在;利用半定量RT-PCR方法对2种转基因藻的hG-CSF转录水平进行比较,发现PsbA启动子驱动效率与Ptac启动子没有明显差异;利用ELISA方法比较hG-CSF蛋白表达量,发现PsbA启动的蓝藻中hG-CSF表达量是Ptac诱导条件下表达量的1.17倍。     

基因工程菌生产rhG-CSF发酵培养基的研究

在摇瓶中对工程菌DH5αpBV220生产rhGCSF的发酵培养基进行了研究,从几种常用的细菌培养基中筛选适合工程菌表达rhGCSF的M9培养基。利用正交试验优化出生产重组人rhGCSF的优化培养基配方,其中优化碳源含量为2%的葡萄糖,氮源为25%的酵母粉、3%的蛋白胨和01%的(NH4)2SO4以及少量的无机盐。使用优化培养基可使rhGCSF的表达量达到338%,比优化前提高了489%。     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

人骨髓细胞培养液中干细胞刺激物的分析研究

人骨髓细胞体外培养液中含有高活力的 CSF,在长期培养过程中,CSF 活力的变化,与 CFU-C 数量的变化有大致平行的趋势。这种 CSF 对狗和小鼠也同样有效。人骨體条件液中的 CSF 对培养中的 CFU-S 也有明显的激发作用。这一结论可以从几个方面获得证据:第一,小鼠骨髓细胞与人骨髓条件液保温六小时后,再测定其中 CFU-S 数,结果是增加了。第二,经亚致死剂量照射的小鼠,腹腔注射适量的人骨髓条件液,其内源性脾结节也明显增多。第三,采用阿糖胞苷自杀的方法,测定小鼠骨髓经与人骨髓条件液保温后,其中 CFU-S 的自杀率也有增高的趋势。上述几方面的实验,说明人骨髓长期培养中存在着某种活性物质,调节体外造血。至于这种物质的来源,以及在体外造血中所起的作用,还需要做很多工作,逐步予以澄清。     

聚乙二醇定点修饰重组人粒细胞集落刺激因子突变体的研究

研究了重组人粒细胞集落刺激因子突变体(rmhG-CSF)的聚乙二醇化修饰、分离纯化和活性鉴定。通过对人重组粒细胞集落刺激因子(rhG-CSF)第1,3,4,5,17位氨基酸进行突变,并在C末端加了一个半胱氨酸,获得了体外活性为原型rhG-CSF150%以上的重组人粒细胞集落刺激因子突变体(rmhG-CSF)。然后用分子量为20kD的甲氧聚乙二醇马来酸酐(mPEG-Mal)修饰rmhG-CSF,反应混合物经离子交换和凝胶过滤柱纯化,得到纯化的聚乙二醇重组人粒细胞集落刺激因子突变体(PEG-rmhG-CSF)。SDS-PAGE电泳分析表明纯化后的PEG-rmhG-CSF的纯度大于95%,体外活性分析表明PEG-rmhG-CSF活性优于目前临床使用的聚乙二醇重组人粒细胞集落刺激因子(PEG-rhG-CSF,NeulastaR○),药代动力学研究表明PEG-rmhG-CSF体内半衰期约为14h,比修饰前延长了7倍。     

聚乙二醇定点修饰重组人粒细胞集落刺激因子突变体的研究

研究了重组人粒细胞集落刺激因子突变体（rmhG-CSF）的聚乙二醇化修饰、分离纯化和活性鉴定。通过对人重组粒细胞集落刺激因子（rhG-CSF）第1,3,4,5,17位氨基酸进行突变,并在C末端加了一个半胱氨酸,获得了体外活性为原型rhG-CSF 150％以上的重组人粒细胞集落刺激因子突变体（rmhG-CSF）。然后用分子量为20kD的甲氧聚乙二醇马来酸酐（mPEG-Mal）修饰rmhG-CSF,反应混合物经离子交换和凝胶过滤柱纯化,得到纯化的聚乙二醇重组人粒细胞集落刺激因子突变体（PEG-rmhG-CSF）。SDS-PAGE电泳分析表明纯化后的PEG-rmhG-CSF的纯度大于95％,体外活性分析表明PEG-rmhG-CSF活性优于目前临床使用的聚乙二醇重组人粒细胞集落刺激因子（PEG-rhG-CSF,Neulasta^?）,药代动力学研究表明PEG-rmhG-CSF体内半衰期约为14h,比修饰前延长了7倍。     

裂变中子照射狗血清集落刺激因子的变化

利用体外半固体琼脂培养骨髓粒单系祖细胞的方法测定狗血清中集落刺激因子(CSF)活力。狗受1.3、1.7和2.0Gy裂变中子照射后1～15d,受照射动物血清CSF持续上升。2.0Gy照射动物死前血清CSF活力可为照射前7～20倍。1.3Gy及1.7Gy照射活存狗照射后13～30d CSF活力直线下降。γ线3.5和2.5Gy照射狗早期血清中CSF活力变动呈波动性,5～10d后持续上升的幅度也不如中子照射动物。 实验结果证明,血清CSF活力和外周血白细胞数的变化呈负相关。本文对中子照射动物血清CSF活力升高的机理和CSF在中子照射动物造血恢复中的意义进行了探讨。     

低能量激光对造血细胞的增殖作用

激光能刺激造血细胞增殖,促使粒-巨噬细胞集落形成单位(GM—CFUc)增多。从人脐带血中分离制备的有核细胞,加入集落刺激因子(CSF)后,形成的GM—CFUc为18.6±12.6/10~4;如果加入的CSF是经激光照射后的细胞制成的,其GM—CFUc为40.5±20.2/10~4;若先用激光照射脐带血的有核细胞,在培养时加入的是未经激光处理细胞制成的CSF,也有增殖效应,GM—CFUc为36.5±18.5/10~4;如果脐带血有核细胞和制备CSF的细胞都用低能激光照射,就可以见到GM—CFUc明显地增多,其值为57.4±41.2/10~4是未经激光处理者的三倍。表明激光照射脐带血有核细胞及制备CSF的细胞均可使GM—CFUc增殖。     

Nanocomplexes of recombinant proteins and polysialic acid: Preparation,characteristics, and biological activity

A preparation of nanocomplexes containing recombinant proteins (interferons α2b and β1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10–20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.     

A proteomics strategy for the enrichment of receptor-associated complexes

Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.     

Tumor necrosis factor-α can induce Langhans-type multinucleated giant cell formation derived from myeloid dendritic cells

The formation of the rich cellular features of MGCs, where the nuclei are arranged circularly at the periphery of the cell (morphologically epithelioid; Langhans-type), is assumed to be associated with any granulomatous disease. The mechanism by which TNF controls the formation of human MGCs in vitro was investigated, focusing on the effect of the TNF-neutralizing antibody. Peripheral blood monocytes were isolated with mAb-coated immunologic magnetic beads and cultured for 10 days in the presence of 20 ng/mL GM-CSF and 10 ng/mL IL-4. These cells were further incubated in the presence of TNF-α with/without its blockade antibodies for 14 days. Myeloid DCs can be generated from peripheral blood monocytes, and both IL-4 and GM-CSF can provide sufficient stimulus for their differentiation. The formation of MGC can be induced in the presence of TNF-α. This reaction was prohibited by the presence of the TNF-neutralizing antibody but not by the presence of anti-TNF receptor II antibody. The activation of Rho and focal adhesion kinases induced by TNF-α stimulation might be linked to cell assembling and the formation of Langhans-type MGCs. MGCs can produce only small amounts of superoxide anions compared to isolated macrophages such as myeloid DCs.     

rhGM-CSF联合氨磷汀治疗放射性口腔黏膜炎的临床疗效及对外周血淋巴细胞亚群的影响

摘要 目的：研究重组人粒细胞/巨噬细胞集落刺激因子（rhGM-CSF）联合氨磷汀治疗放射性口腔黏膜炎患者的临床治疗疗效以及对外周血淋巴细胞亚群的影响。方法：选取2019年1月到2020年12月在我院接收治疗的放射性口腔黏膜炎患者60例,随机分为对照组和rhGM-CSF组两组,每组30例。对照组接受静脉滴注氨磷汀治疗,rhGM-CSF组接受rhGM-CSF漱口液漱口联合静脉滴注氨磷汀治疗。比较两组患者临床治疗疗效、治疗后口腔黏膜炎评分、疼痛评分、吞咽功能、血清炎症因子以及外周血淋巴细胞亚型。结果：（1）rhGM-CSF组患者临床治愈率和治疗总有效率分别为26.67 %和90.00 %,均高于对照组6.67 %临床治愈率和70.00 %临床治疗总有效率（P<0.05）。（2）治疗后,rhGM-CSF组口腔黏膜炎评分、疼痛评分、吞咽功能、血清干扰素-γ（IFN-γ）、肿瘤坏死因子（TNF-α）和白介素-6（IL-6）含量均显著低于对照组患者（P<0.05）。（3）rhGM-CSF组患者治疗后外周血CD3⁺T淋巴细胞与对照组比较无差异（P>0.05）,外周血CD4⁺和CD4⁺/CD8⁺T淋巴细胞亚群显著高于对照组患者,而CD8⁺T淋巴细胞亚群显著低于对照组患者（P<0.05）。结论：rhGM-CSF漱口液联合静脉滴注氨磷汀治疗治疗放射性口腔黏膜炎临床疗效更优,有助于减轻患者外周血炎症反应,增强细胞免疫功能。     

小剂量rhGM-CSF化疗前应用的临床研究

目的:探讨小剂量重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)化疗前应用在减轻骨髓抑制、缩短住院时间、减少医疗费用方面的作用。方法:实验组化疗前48小时GM-CSF300μg皮下注射1次,出现骨髓抑制后实验组与对照组均给予rhGM-CSF300μg皮下注,1次/日,直至WBC≥4.0×109/L。结果:实验组的白细胞减少、粒细胞减少及血小板下降均较对照组轻,(P0.05),实验组白细胞恢复时间短于对照组,(P0.05),住院天数及药费也存在显著性差异(P0.05)。结论:化疗前给予rhGM-CSF,可以有效地降低骨髓抑制的发生率及发生程度,缩短骨髓恢复时间,在降低医疗费用、加快床位周转、提高病床使用率方面也显示出一定的优势。