重组人酸性成纤维细胞生长因子突变体的聚乙二醇定点修饰研究.docVIP

中文摘要 蛋白质药物由于具有作用专一、高效等特点，在人类健康中发挥着日益重要的作用。蛋白质药物的疗效不仅取决于蛋白质特定的化学结构，而且还取决于其特定的空间结构，因此可以通过基因工程、化学修饰等手段来改变蛋白质的结构，从而部分甚至全部解决蛋白质药物的缺陷（如稳定性差、易被循环系统快速清楚从而造成体内循环半衰期很短、存在免疫原性和抗原性等）。其中化学修饰以其灵活多样、简便易行的特点而得到广泛的应用。 人酸性成纤维细胞生长因子是一类来源于中胚层和神经外胚层的多种类型的细胞具有广泛生物学活性的细胞生长因子，早在1960年，人们就已发现并确认其是一种介导细胞间信号传递的多肽类物质。经过近半个世纪的研究和临床实践，发现该因子在维持人体平衡和治疗诸如各类体表创伤、溃疡以及心肌和神经组织损伤等方面起着至关重要的作用。但是，长期以来，人酸性成纤维细胞生长因子的体内稳定性差、生物半衰期短、易发生免疫应答反应等缺陷也大大限制了其临床应用。 本文根据文献方法合成了巯基PEG化试剂-mPEG马来酸酰亚胺活化酯，并采用基因工程技术将haFGF98、132位的半胱氨酸突变为丝氨酸，使其仅保留31位的半胱氨酸。在此基础上，采用上述修饰剂对获得的haFGF突变体进行了定点化学修饰，并对修饰产物的分离制备、活性、稳定性、免疫反应性和免疫原性、药动学特点以及对大鼠缺血再灌注损伤的保护作用作了较为系统的研究，得到了较为理想的结果，从而为改善和提高生长因子及其他蛋白药物的生物学性能提供了有效的理论和实验手段，具有较为重要的理论和现实意义。 关键词：人酸性成纤维细胞生长因子 聚乙二醇 化学修饰 Abstract Protein drugs hold great promise as therapeutic agents for their high specificity and efficacy. The efficacy of protein drugs not only depends on the chemical structure, but also on their three-dimensional structures. Many of the protein drugs are readily degraded by proteolytic enzymes, cleared rapidly by kidneys. In addition, they generate neutralizing antibodies and have a short circulating half-life. These shortcomings can be partially or even totally overcomed by methods of gene engineering and chemical modification. Chemical modification has been widely used for its flexibility and simplicity. Human acid fibroblast growth factor (haFGF) is a cytokine, which has broad-spectrum biological effect on many type cells of mesodermal and neuroectodermal origin. haFGF has been recognized as a peptide to mediate the signal transduction. As an efficient tissue repairing stimulator and a cardio- and neuro-protector, haFGF has potentials in wound healing, bone damage curing and cardio- and neuro-diseases treatment. But some disadvantages of aFGF such as low stability, short half life in vivo and strong immunogenic activity, limited its clinical applications. In this work, the activated polyethylene glycol-maleimide was synthe