發(fā)布時間：2018-02-22 21:30

  本文關(guān)鍵詞： 肝素 粒細胞集落刺激因子 細胞 相互作用 毛細管電泳　出處：《中國科學(xué)院研究生院（大連化學(xué)物理研究所）》2006年博士論文　論文類型：學(xué)位論文

【摘要】： 肝素等硫酸多糖是一類重要的生物分子,它通過與蛋白質(zhì)相互作用在許多生物學(xué)過程中起著重要作用,如信號傳導(dǎo)、細胞生長與分化等。研究此類相互作用對深入理解生物和生理過程有重要意義,并對以糖為基礎(chǔ)的新藥研發(fā)有促進作用。本文以毛細管電泳(CE)為主要技術(shù)平臺,結(jié)合高效液相色譜(HPLC)、質(zhì)譜(MS)和流式細胞術(shù)等系統(tǒng)研究了肝素等硫酸多糖與粒細胞集落刺激因子(G-CSF)等蛋白質(zhì)的相互作用。 利用毛細管區(qū)帶電泳(CZE)研究了不同類型蛋白質(zhì)與肝素的相互作用。發(fā)現(xiàn)CZE既可研究動力學(xué)上慢速解離平衡體系,又可研究快速解離平衡體系,擴展了CZE的應(yīng)用范圍。發(fā)現(xiàn)粒細胞巨嗜細胞集落刺激因子與肝素的相互作用具有特異性,并首次測定了結(jié)合常數(shù)。 利用CZE系統(tǒng)研究了不同分子量、不同來源及不同硫酸化位點的肝素和G-CSF的相互作用,進行了定性和定量測定。首次發(fā)現(xiàn)G-CSF能和肝素結(jié)合,且結(jié)合的強弱與肝素的鏈長、來源及硫酸化位點有關(guān)。發(fā)展了一種既適于分離肝素寡糖、又適于研究肝素與蛋白質(zhì)相互作用的方法。首次在肝素寡糖分離基礎(chǔ)上研究其與蛋白質(zhì)的相互作用,并找到了與G-CSF結(jié)合的組分。該方法勿須加入任何添加劑,即可在正向分離模式下使一種肝素酶降解肝素所得寡糖快速達到基線分離。對該寡糖進行了定性研究,未發(fā)現(xiàn)三硫酸化二糖,表明所用肝素酶可選擇性的降解肝素。用HPLC和MS對該寡糖進行進一步分離和鑒定,得出了各肝素二糖的分子量,與CE所得結(jié)果一致。 作為肝素的擴充,研究了其它硫酸聚糖和G-CSF的相互作用,并對硫酸聚糖的結(jié)構(gòu)對相互作用的影響進行了定性定量考察。發(fā)現(xiàn)糖鏈長度和硫酸化程度是其與G-CSF相互作用的關(guān)鍵因素。 在上述分子水平研究的基礎(chǔ)上,將G-CSF與其結(jié)合多糖作用于G-CSF依賴鼠髓白血病細胞株,NFS-60。用流式細胞術(shù)等多種生物學(xué)方法研究了作用后細胞的增值、分化及細胞周期變化等。首次發(fā)現(xiàn)了三種硫酸多糖能顯著抑制NFS-60細胞生長并誘導(dǎo)其分化為成熟細胞。
[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.
【學(xué)位授予單位】：中國科學(xué)院研究生院（大連化學(xué)物理研究所）
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R341

【引證文獻】

相關(guān)期刊論文 前1條

1 陳思群;孫自才;陳建軍;陳曉暉;;糖-蛋白質(zhì)相互作用在酶固定及蛋白質(zhì)識別與分離中的應(yīng)用[J];中國生物工程雜志;2012年04期

相關(guān)碩士學(xué)位論文 前1條

1 吳艷芳;低分子量肝素精細結(jié)構(gòu)的分析研究[D];東華大學(xué);2012年

，

本文編號：1525353