含RGD肽CP9修饰的聚乙烯亚胺复合物的理化特性及其转基因功能的研究  

Physicochemical characters and gene delivery function of polyethy-lenimine modified with peptide CP9 containing RGD sequence

作　　者：李达[1] 王青青[1] 汤谷平[2] 李经忠[1] 黄宏靓[1] 沈芬平[1] 余海[1] 

机构地区：[1]浙江大学免疫学研究所,浙江杭州310058 [2]浙江大学化学生物和药物化学研究所,浙江杭州310028

出　　处：《浙江大学学报（医学版）》2006年第6期615-621,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家高技术研究发展计划(863)资助项目(2003AA216041);国家重点基础研究规划(973)资助项目(2004CB518802)

摘　　要：目的:构建含精氨酸-甘氨酸-天冬氨酸(RGD)肽CP9修饰的聚乙烯亚胺(PE I),观察其理化特性和转基因功能。方法:通过琥珀酰亚胺-3-(2-嘧啶二硫)丙酸酯(N-Succin im idy l-3-(2-pyridy ld ith io)]prop ionate,SPDP)将CP9偶联到PE I上,合成新型转基因载体CP9-PE I。用1H-NMR和FT-IR验证CP9的偶联;用凝胶电泳阻滞实验、电镜和粒径检测,观察CP9-PE I浓缩质粒DNA的能力以及浓缩质粒DNA后形成的转染颗粒形态和粒径;通过CP9-PE I在人肝癌细胞株H epG 2中的转染实验来验证CP9对PE I的转染效率的影响;通过游离CP9肽的竞争抑制实验验证CP9-PE I的整合素靶向功能。结果:CP9成功偶联到PE I上;CP9-PE I能有效浓缩质粒DNA,形成的转染颗粒形态为圆形或类圆形,在N/P比为10时,粒径约为200 nm。CP9-PE I的转染效率是PE I的2倍,游离CP9肽能抑制CP9-PE I的转染效率。结论:修饰了CP9的PE I能有效增加转染效率,具有整合素的靶向能力,是一种具有应用前景的转基因载体。Objective： To construct a novel gene delivery vector using polyethylenimine （PEI） as backbone modified with the peptide CP9 containing Arginine-Glycine-Aspartic acid （RGD） sequence and to verify its physicochemical characters and the gene delivery function. Methods： The chemical linker [N-Succinimidyl-3-（2-pyridyldithio）] propionate （SPDP） was employed to bind CP9 onto PEI to form a novel gene delivery vector CP9-PEI. The ^1H-NMR and FT-IR were used to verify the linkage of CP9. The plasmid DNA condensing ability of CP9-PEI, the shape and the particle size of the polyplexes formed with CP9-PEI-plasmid DNA were demonstrated by gel retardation assay, electron microscope observation and particle size assay, respectively. The enhanced transfection efficiency and the integrin targeting capacity were detected by the transfection experiments in HepG2 cells and free RGD peptide inhibition test. Results： CP9 was linked onto PEI successfully. The new synthesized vector CP9-PEI could efficiently condense plasmid DNA at N/P ratio of 4 and when N/P ratio was equal to 10, the shape of polyplexes formed with CP9-PEI-plasmid DNA was round or round-alike with particle size of about 200 nm. The transfection efficiency of CP9-PEI was nearly 2 times of PEI in HepG2 cells and the free RGD peptide had the inhibition effect on the efficiency of CP9-PEI. Conclusion. The modification of CP9 on PEI can improve the transfection efficiency of PEI and has the integrin targeting ability.

关 键 词：聚乙烯亚胺 转基因 遗传载体 精氨酸 甘氨酸 天冬氨酸 RGD 

分 类 号：R346[医药卫生—基础医学]