肝素等硫酸多糖与粒细胞集落刺激因子等蛋白质在分子和细胞水平上的相互作用研究

发布时间：2018-02-22 21:30

本文关键词： 肝素粒细胞集落刺激因子细胞相互作用毛细管电泳　出处：《中国科学院研究生院（大连化学物理研究所）》2006年博士论文　论文类型：学位论文

【摘要】： 肝素等硫酸多糖是一类重要的生物分子,它通过与蛋白质相互作用在许多生物学过程中起着重要作用,如信号传导、细胞生长与分化等。研究此类相互作用对深入理解生物和生理过程有重要意义,并对以糖为基础的新药研发有促进作用。本文以毛细管电泳(CE)为主要技术平台,结合高效液相色谱(HPLC)、质谱(MS)和流式细胞术等系统研究了肝素等硫酸多糖与粒细胞集落刺激因子(G-CSF)等蛋白质的相互作用。利用毛细管区带电泳(CZE)研究了不同类型蛋白质与肝素的相互作用。发现CZE既可研究动力学上慢速解离平衡体系,又可研究快速解离平衡体系,扩展了CZE的应用范围。发现粒细胞巨嗜细胞集落刺激因子与肝素的相互作用具有特异性,并首次测定了结合常数。利用CZE系统研究了不同分子量、不同来源及不同硫酸化位点的肝素和G-CSF的相互作用,进行了定性和定量测定。首次发现G-CSF能和肝素结合,且结合的强弱与肝素的链长、来源及硫酸化位点有关。发展了一种既适于分离肝素寡糖、又适于研究肝素与蛋白质相互作用的方法。首次在肝素寡糖分离基础上研究其与蛋白质的相互作用,并找到了与G-CSF结合的组分。该方法勿须加入任何添加剂,即可在正向分离模式下使一种肝素酶降解肝素所得寡糖快速达到基线分离。对该寡糖进行了定性研究,未发现三硫酸化二糖,表明所用肝素酶可选择性的降解肝素。用HPLC和MS对该寡糖进行进一步分离和鉴定,得出了各肝素二糖的分子量,与CE所得结果一致。作为肝素的扩充,研究了其它硫酸聚糖和G-CSF的相互作用,并对硫酸聚糖的结构对相互作用的影响进行了定性定量考察。发现糖链长度和硫酸化程度是其与G-CSF相互作用的关键因素。在上述分子水平研究的基础上,将G-CSF与其结合多糖作用于G-CSF依赖鼠髓白血病细胞株,NFS-60。用流式细胞术等多种生物学方法研究了作用后细胞的增值、分化及细胞周期变化等。首次发现了三种硫酸多糖能显著抑制NFS-60细胞生长并诱导其分化为成熟细胞。
[Abstract]:Sulfated polysaccharides such as heparin are important biomolecules that play an important role in many biological processes, such as signal transduction, by interacting with proteins. Cell growth and differentiation. The study of this kind of interaction is of great significance in understanding biological and physiological processes and promoting the development of new drugs based on sugar. In this paper, capillary electrophoresis (CEE) is used as the main technical platform. The interaction between heparin sulfate polysaccharides and granulocyte colony stimulating factor (G-CSF) was studied by means of high performance liquid chromatography (HPLC) HPLC- (MS) and flow cytometry (FCM). The interaction of different proteins with heparin was studied by capillary zone electrophoresis (CZE). It was found that CZE could be used to study both the slow dissociation equilibrium system and the fast dissociation equilibrium system. It was found that the interaction between granulocyte giant eosinophil colony stimulating factor and heparin was specific, and the binding constant was determined for the first time. The interaction of heparin and G-CSF with different molecular weight, different sources and different sulfated sites was studied by CZE system. It was first found that G-CSF could bind to heparin, and the binding strength was associated with the chain length of heparin. A method was developed for the separation of heparin oligosaccharides and for the study of the interaction between heparin and protein. The interaction between heparin oligosaccharide and protein was studied for the first time on the basis of separation of heparin oligosaccharides. The composition combined with G-CSF was found. Without adding any additive, the oligosaccharides obtained from the degradation of heparin by a heparinase could reach the baseline separation quickly without adding any additive. The qualitative study of the oligosaccharides was carried out. No trisulfated disaccharide was found, which indicated that heparin could be selectively degraded by heparinase. The molecular weight of each heparin disaccharide was obtained by further separation and identification by HPLC and MS, which was consistent with the result obtained by CE. As an expansion of heparin, the interaction between other sulfans and G-CSF was studied. The effects of the structure of sulfate on the interaction were investigated qualitatively and quantitatively. It was found that the length of sugar chain and the degree of sulfation were the key factors for the interaction between glycomannan and G-CSF. On the basis of the above molecular studies, G-CSF and its binding polysaccharides were applied to G-CSF dependent rat myeloid leukemia cell line NFS-60. flow cytometry and other biological methods were used to study the proliferation of the treated cells. It is the first time to find that three kinds of sulfated polysaccharides can significantly inhibit the growth of NFS-60 cells and induce them to differentiate into mature cells.
【学位授予单位】：中国科学院研究生院（大连化学物理研究所）
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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