体外定点糖基化修饰的内皮抑素活性短肽靶向性抗新生血管生成与抗肿瘤作用机制研究

项目名称： 体外定点糖基化修饰的内皮抑素活性短肽靶向性抗新生血管生成与抗肿瘤作用机制研究

项目编号： No.81473129

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 谭海宁

作者单位： 山东大学

项目金额： 60万元

中文摘要： 以内皮抑素的两个活性肽段为研究对象，以具抗肿瘤生长和转移活性、具靶向性且结构明确的肝素寡糖、壳聚糖和甘露五糖为修饰剂，体外实现定点糖基化修饰；评价产物抗新生血管生成作用和体内对小鼠移植性实体瘤生长抑制作用，采用流式细胞术、激光共聚焦显微镜技术和放射性同位素标记技术，研究体内外的细胞靶向性及机制；研究对小鼠肝癌转移株细胞与冰冻淋巴结切片体外黏附和体内淋巴道转移的影响，ELISA法研究与L-选择素、甘露糖受体的结合情况，分析抗肿瘤淋巴道转移的机制；同时用Western blot和RT-PCR等技术研究对实体瘤组织中血管内皮细胞生长因子（VEGF）和基质金属蛋白酶（MMP）等相关因子表达的影响，分析抗肿瘤机制；最后，采用放射性标记的方式进行修饰产物体内半衰期等药代动力学和组织分布研究。本项目有助于突破蛋白质或多肽的体外定点糖基化修饰技术，评价定点糖基化修饰的ES短肽的靶向性抗肿瘤增殖及转移机制。

中文关键词： 抗肿瘤；内皮抑素短肽；体外定点糖基化修饰；抗血管生成；作用机制

英文摘要： In order to overcome the disadvantages of endostatin (ES) fragments, heparin oligosaccharides, N,N,N-trimethyl chitosan chloride (TMC) and unsymmetrical branched Man5 oligosaccharide will be used to modify two different ES fragments. After modification, the antiangiogenesis and anti-tumor activity will be studied in several models. Besides that, Flow cytometry and confocal laser microscope will also be used to study the intracellular localization of modified ES fragments. Also, the effects of modified ES fragments on mice hepatocellular carcinoma cell lines both in vitro and in vio will be studied; ELISA will be employed to research the competing inhibition on binding of L-selectin with L-selectin receptors or mannose receptors. Meanwhile, western blotting and RT-PCR will be used to study the action mechanism by detect the expression of VEGF and MMP in tumor tissue. At last, the modified ES fragements will be radio labeled, and then the pharmacokinetics and tissue distribution will be studied. The carry out of this project will be useful on breaking the current limitation of protein or peptide site specific modification methods, and will be meaningful if the inhibition on tumor proliferation and migration mechanism of modified ES fragments could be totally researched.

英文关键词： Anti-tumor;endostatin fragements;specific glycosylation modification in vitro;antiangiogenesis;action mechanishm