狂犬疫苗单克隆抗体制备过程中细胞融合条件的探索

在充分了解SP2/0细胞系的生长、增殖和遗传特性后,以聚乙二醇(PEG)诱导动物细胞融合为契机,意在探索不同分子量、不同浓度的PEG作融合剂对诱导SP2/0细胞与脾细胞融合的最适条件,在HAT选择培养基下通过细胞融合率的变化进行比较。结果表明,分子量为4000,浓度为50%的PEG诱导的细胞融合率最高,为下一步制备狂犬病毒疫苗单克隆抗体奠定基础。     

聚乙二醇诱导鸡红细胞融合条件的优化

目的:探讨聚乙二醇诱导鸡红细胞融合最适条件.方法:以鸡红细胞为材料,聚乙二醇为诱导剂,从细胞密度、融合温度、聚乙二醇浓度及反应时间进行单因素实验,并设计3因素3水平的正交实验计算细胞融合率.结果:鸡红细胞融合的最佳条件是:细胞密度为2×106个/ml,聚乙二醇浓度为35%,反应温度为35℃,反应时间40min.结论:在该条件下,细胞融合率最高达33.06%.     

Mg2+浓度对细胞融合效果的影响

目的:细胞融合是细胞生物学中一种常用的技术,有着广泛的应用,如单克隆抗体制备,核质研究,疫苗研发等.其中,聚乙二醇(PEG)化学融合是最为常用的一种细胞融合技术,影响PEG化学细胞融合效果的因素有很多,但是对一些具体因素的研究的不是很全面.本文旨在为了更全面的了解PEG诱导的化学细胞融合的影响因素,优化融合条件,以此扩大PEG化学融合应用范围.方法:以鸡血血红细胞为材料,通过调节已有的Hanks融合液中镁离子浓度,比较各实验组以及对照组的细胞融合率,探究了Mg2+浓度对细胞融合效果的影响,确定了为提高细胞融合效率应使用的Mg2+浓度区间.结果:可以在原有的Hanks配方的基础上,调节Mg2+浓度至10 mmol/L-20 mmol/L这个范围,细胞融合率较大.结论:Mg2+对细胞融合有一定影响,通过调节Mg2+浓度至上述合适区间可以达到较高的细胞融合率,从而为PEG化学融合提供了一种优化方案.     

三氯生单独暴露对斑马鱼幼鱼眼部的毒性研究

三氯生(2,4,4’-三氯均二苯脲, triclosan, TCS)是环境中应用最为广泛的新兴外源性污染物之一,可强烈地吸附在土壤、沉积物、胶体中。TCS具有生物蓄积作用,在生物体内的富集浓度远超过在水、土壤等环境中的浓度,并且可通过生物富集作用进入食物链。本实验以斑马鱼(Danio rerio)胚胎为模型,探讨不同浓度的TCS暴露对斑马鱼胚胎、幼鱼发育的影响。结果发现, TCS暴露对斑马鱼胚胎和幼鱼具有较强的致畸、致死效应。为了进一步了解TCS对斑马鱼眼部超微结构的影响,将其眼部电镜切片放在透射电镜下观察。结果显示, TCS暴露后斑马鱼幼鱼眼部感光细胞外节盘数量明显减少,外网状层及内网状层较对照组明显变薄且空泡化严重,外核层细胞核固缩现象严重甚至部分细胞核出现溶解的现象。实验结果表明TCS暴露对斑马鱼幼鱼眼部发育有潜在的毒性作用,这可为进一步探讨TCS对人体潜在的毒性作用提供科学、可靠的依据。     

葡萄糖浓度对细胞融合效果的影响

目的:细胞融合是细胞生物学领域近30年来得到迅速发展的一项新兴技术手段,因其操作简便、人工可控等优点在研究核质互作、肿瘤发生、疫苗研发和培育新型生物品种等方面均有广泛应用。其中,利用聚乙二醇(PEG)进行化学融合是细胞融合中最为常用且简便的技术手段。PEG化学融合效果受到多种因素影响,如PEG浓度、Ca2+、Mg2+、pH值等,然而对于糖类物质在细胞融合中的影响未见报道。本文旨在为了更全面了解PEG法诱导的化学细胞融合,通过优化融合条件以提高化学细胞融合效率。方法:选取鸡血血细胞为材料,通过改变原Hanks缓冲液中葡萄糖浓度,观察比较各组细胞融合率,探究葡萄糖浓度在化学细胞融合中的影响,并通过对比结果获得了对于鸡血血细胞应采用的最适葡萄糖浓度区间。结果:对于鸡血血细胞融合实验,葡萄糖浓度在10-14 mmol/L范围内细胞融合效率较原Hanks液配方高2倍左右。结论:葡萄糖对细胞融合效果具有一定的影响,可以通过调节葡萄糖浓度提高细胞融合率,从而为PEG化学细胞融合提供一种更为优化的方案。     

Mg^2＋浓度对细胞融合效果的影响

目的：细胞融合是细胞生物学中一种常用的技术,有着广泛的应用,如单克隆抗体制备,核质研究,疫苗研发等。其中,聚乙二醇（PEG）化学融合是最为常用的一种细胞融合技术,影响PEG化学细胞融合效果的因素有很多,但是对一些具体因素的研究的不是很全面。本文旨在为了更全面的了解PEG诱导的化学细胞融合的影响因素,优化融合条件,以此扩大PEG化学融合应用范围。方法：以鸡血血红细胞为材料,通过调节已有的Hanks融合液中镁离子浓度,比较各实验组以及对照组的细胞融合率,探究了Mg^2＋浓度对细胞融合效果的影响,确定了为提高细胞融合效率应使用的Mg^2＋浓度区间。结果：可以在原有的Hanks配方的基础上,调节Mg^2＋浓度至10 mmol/L-20 mmol/L这个范围,细胞融合率较大。结论：Mg^2＋对细胞融合有一定影响,通过调节Mg^2＋浓度至上述合适区间可以达到较高的细胞融合率,从而为PEG化学融合提供了一种优化方案。     

雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

为探索药用植物雷公藤(Tripterygium wilfordii)悬浮细胞原生质体提取的最优条件,并建立雷公藤原生质体瞬时转化体系,以雷公藤悬浮细胞为材料,对酶解液配比、酶解时间、甘露醇浓度及处理转速进行考察。用PEG介导的瞬时转化法将外源基因转化到雷公藤原生质体中。结果表明,以雷公藤悬浮细胞为材料提取原生质体的最佳条件是酶液配比为2.0%纤维素酶+0.5%果胶酶+0.5%离析酶,甘露醇浓度为0.6 mol·L–1,酶解10小时,处理转速为67×g;用PEG介导法将含有编码GFP的植物表达载体转化雷公藤悬浮细胞原生质体,激光共聚焦扫描显微镜下细胞显示绿色荧光。通过实验筛选得到雷公藤悬浮细胞原生质体的最佳提取条件,建立了雷公藤悬浮细胞原生质体的瞬时转化体系,为进一步开展雷公藤功能基因及合成生物学研究奠定了基础。     

CHO细胞表达系统研究新进展

中国仓鼠卵巢细胞(ChineseHamsterOvaryCell)是目前重组糖基蛋白生产的首选体系。本文综述近几年来该系统在生产基因工程药物方面的研究进展、存在问题和发展方向。     

应用活体骨髓瘤细胞制备单克隆抗体

【目的】用活体骨髓瘤细胞SP2/0做融合提高融合率制备单克隆抗体,并与常规方法比较效果。【方法】将SP2/0细胞打到8周龄的SPF级BALB/c小鼠皮下,待实体瘤生长到直径达2～3cm时无菌解剖取实体瘤,分离出骨髓瘤细胞进行融合。同时用培养基培养SP2/0细胞进行融合做比较,分两组进行。比较两种方法的融合率以及两种方法制备出来的单克隆抗体的相对亲和力。【结果】做了6次融合,实体瘤融合组融合率为70.4％,常规法融合组44.6％,两种方法制备单抗的相对亲和力均达到1:100000以上。【结论】利用活体实体瘤细胞进行融合能明显提高细胞融合率。     

草鱼细胞融合及早熟凝集染色体的诱导

用聚乙二醇（PEG）诱导草鱼ZC-7901细胞株融合,测定了不同浓度的PEG诱导草鱼组胞的融合率,从而得出了PEG诱导草鱼细胞融合的适宜浓度为45%（W/W）。用45%PEG诱导间期细胞（I期细胞）与分裂期细胞（M期细胞）融合,早熟凝集染色体（PCC）的诱导率是18.85%。观察PCC形态,G1-PCC呈单股染色体纤维形;G2-PCC呈双股染色纤维形,较分裂期细胞染色体细长;S-PCC呈“粉末状”染色体片段。     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

Enhancement of the recognition by cytotoxic T lymphocytes (CTL) of target membrane antigens after fusion with whole cells

Plasma membrane vesicles (R4-PM) prepared from mouse lymphoma cells (RDM4,H2k) were employed to investigate requirements for recognition of target cell membranes by allogeneic cytotoxic T lymphocytes (CTL). Using immunofluorescent staining and fluorescence microscopy, the R4-PM were tested for binding to CTL and were found to bind to these effector cells in a specific manner. However, this binding was very inefficient compared to the binding of whole RDM4 cells to CTL. The R4-PM were then attached to P388D1 cells (H-2d) in the presence of wheat germ agglutinin and polyethylene glycol (PEG), both under conditions which promote membrane fusion (40% PEG) and under conditions which do not (10% PEG). About 1 cell equivalent R4-PM becomes associated per P388D1 cell in both situations. In the cytotoxicity assays that were carried out, the P388D1 cells which had R4-PM attached under fusion conditions were lysed by CTL directed against H2k in a specific manner, while the P388D1 cells which had R4-PM attached under nonfusion conditions were not lysed above background levels by these CTL. These results suggest that recognition of target cells by allogeneic CTL such that lysis occurs requires more than presentation of the alloantigens as they are expressed in plasma membrane vesicles. However, fusion of these vesicles back into living cells apparently enhances the ability of the alloantigens to be recognized.     

Fluorescence microscopic study of cells and polykaryons in the early periods following polyethylene glycol treatment

A fluorescence-microscopical study is made of cultured murine fibroblasts (L-cells) in early periods after the treatment with polyethylene glycol (PEG). Optimal conditions of fusion procedure were found under which the effectiveness of fusion was the highest and the toxical effect of PEG the lowest. The number of dead cells after the treatment with PEG did not exceed 10%. No significant changes in chromatin cytochemical properties (Acridine Orange and Olivomycin binding) were observed in the early periods of PEG treatment, that allows to use PEG for studying chromatin properties in hybrid cells obtained by PEG fusion. By means of PEG fusion, the hybrid cells with prematurely condensed chromosomes and also hybrids between animal and yeast cells have been obtained.     

Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non-genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by testing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect genotoxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage.     

Cell fusion and intramembrane particle distribution in polyethylene glycol-resistant cells

The distribution of intramembrane particles (IMP) as revealed by freeze- fracture electron microscopy has been analyzed following treatment of mouse L cells and fusion-deficient L cell derivatives with several concentrations of polyethylene glycol (PEG). In cell cultures treated with concentrations of PEG below the critical level for fusion, no aggregation of IMP was observed. When confluent cultures of the parental cells are treated with 50% PEG, greater than 90% of the cells fuse, and cold-induced IMP aggregation is extensive. In contrast, identical treatment of fusion-deficient cell lines shows neither extensive fusion nor IMP redistribution. At higher concentrations of PEG, however, the PEG-resistant cells fuse extensively and IMP aggregation is evident. Thus the decreased ability of the fusion- deficient cells to fuse after treatment with PEG is correlated with the failure of IMP aggregation to occur. A technique for quantifying particle distribution was developed that is practical for the accurate analysis of a large number of micrographs. The variance from the mean number of particles in randomly chosen areas of fixed size was calculated for each cell line at each concentration of PEG. Statistical analysis confirms visual observation of highly aggregated IMP, and allows detection of low levels of aggregation in parental cells that were less extensively fused by exposure to lower concentrations of PEG. When low levels of fusion were induced in fusion-deficient cells, however, no IMP aggregation could be detected.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Cell fusion between temperature-sensitive mutants of a Drosophila melanogaster cell line.

Temperature-sensitive (ts) mutants were isolated in a cell line of Drosophila melanogaster, GM1, by ethyl methanesulfate treatment. Two of them, ts15 and ts58, formed colonies at 23 degrees C but not at 30 degrees when inoculated at densities of/or less than 10(5) cells per 60 X 15-mm dish. By using these ts mutants, cell fusion was attempted with polyethylene glycol (PEG) 6000. Several colonies per dish developed at 30 degrees C when different ts mutants were mixed, treated with PEG, and inoculated at a density of 10(4) cells per dish. Cells in some of the colonies thus developed were propagated and their temperature-sensitive character and karyotypes were studied. The results indicated that cell fusion could be induced with PEG and that the cells which formed colonies at 30 degrees C after PEG treatment were the hybrids in which the temperature-sensitive lesions in the mutants were complemented.     

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines. Revisions requested 27 October 2005; Revisions received 12 December 2005