高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (08): 1757-1764.doi: 10.3969/j.issn.0251-0790.2012.08.023

• 生物化学 • 上一篇    下一篇

普鲁兰多糖为骨架的新型阳离子非病毒基因载体

王静云, 窦柏蕊, 包永明, 李森武   

  1. 大连理工大学生命科学与技术学院, 大连 116024
  • 收稿日期:2011-09-26 出版日期:2012-08-10 发布日期:2012-08-10
  • 通讯作者: 王静云, 女, 博士, 副教授, 博士生导师, 主要从事酶工程和生物材料研究. E-mail: wangjingyun67@126.com; wangjingyun67@dlut.edu.cn E-mail:wangjingyun67@126.com
  • 基金资助:

    国家自然科学基金(批准号: 20876024)资助.

Novel Pullulan-based Non-virus Cationic Gene Delivery Vector

WANG Jing-Yun, DOU Bai-Rui, BAO Yong-Ming, LI Sen-Wu   

  1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
  • Received:2011-09-26 Online:2012-08-10 Published:2012-08-10

摘要: 通过琥珀酸酐将低分子量支化聚乙烯亚胺(PEI, 分子量1000)偶联到普鲁兰多糖(Pullulan)上, 合成了新型基因载体P-PEI. 利用 1H NMR、 FTIR、 粒度仪、 Zeta电位仪、 透射电镜和凝胶电泳对聚阳离子载体及其与质粒pDNA 的复合物进行了表征. 凝胶阻滞实验结果证明, 载体P-PEI在体外可以通过静电相互作用稳定结合pDNA, 并能有效抑制DNA水解酶及血清成分对pDNA的降解. 噻唑蓝(MTT)细胞毒性测试、 绿色荧光蛋白表达质粒(pGFP)及荧光素酶表达质粒(pGL3)转染实验结果表明, 载体P-PEI在N/P高达12.5时对细胞MCF-7, HeLa和COS-7的毒性低于PEI; 当N/P 为6.25时能有效将pGFP和pGL3带入Hela 细胞并表达, 最佳转染效率及荧光素酶活分别为, 比Lipo 2000[(49.13±0.61)%, (58.47±7.62)×108 RLU/mg蛋白) 略低. 因此以Pullulan为骨架材料的P-PEI是一种新的有潜在应用价值的非病毒基因载体.

关键词: 普鲁兰多糖, 聚乙烯亚胺, 绿色荧光蛋白表达质粒pGFP, 非病毒基因载体

Abstract: A novel non-virus gene delivery vector(P-PEI) was synthesized by grafting low-molecular branched PEI(molecular weight: 1000) onto the backbone of pullulan with succinic acid as a spacer. 1H NMR, FTIR, particle size analysis and zeta-potential measurements, TEM and gel electrophoresis were performed to characterize P-PEI and P-PEI/pDNA complexes. Gel electrophoresis retardation assay demonstrated that P-PEI can wrap pDNA with electrostatic interaction excellently even in the presence of heparin, and protect pDNA from the degradation by DNase I and serum. Compared with PEI/pDNA complexes, the P-PEI/pDNA complexes show relative lower cytotoxicity against MCF-7, HeLa and COS-7 cells, even at high N/P(up to 12.5) demonstrated by MTT assay. Flowcytometry revealed that the highest transfection efficiency for P-PEI as a delivery agent was obtained at N/P ratio of 6.25, which was comparable with that of Lipofectamine 2000, and consistent with the results of luciferase expression. These results suggest that the pullulan-grafted branched polyethylenimine polymer (P-PEI) might be an excellent biocompatible candidate for gene delivery systems.

Key words: Pullulan, Polyethylenimine, Expression plasmid for green fluorescent portein pGFP, Non-virus gene vector

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