新合成壳聚糖季铵盐/DNA复合物在Hela细胞中的转染效率

背景:壳聚糖季铵盐衍生物可以提高载体的转染效率,同时解决了壳聚糖溶解性差的问题,扩展了其pH值适用范围.目的:合成一种新型壳聚糖季铵盐载体,研究其与质粒结合的条件及转染效率,并与壳聚糖进行对比.设计、时间及单位:分子生物学,对比观察实验,于2008-08/10在浙江大学医学院附属第二医院完成.材料:壳聚糖季铵盐-N-2-羟丙基三甲基氯化铵壳聚糖为北京理工大学高分子材料实验室合成.质粒pEGFP-C1为浙江大学医学院郑一春惠赠.Hela细胞为浙江大学医学院程静提供.方法:将N-2-羟丙基三甲基氯化铵壳聚糖配制成质量浓度为0.2 g/L、pH值为5.5(或6.9、7.6)、乙酸钠终浓度为50 mmol/L的乙酸溶液,迅速与质粒pEGFP-C1混合,并在旋转混合器上涡流15～30 S,室温静置30 min以上,促进复合物的形成.主要观察指标:通过凝胶阻滞实验研究了pH值及时间对壳聚糖季铵盐与质粒结合能力的影响.通过荧光倒置显微镜观察壳聚糖季铵盐作为载体转染Hela细胞的效率,并与壳聚糖进行对比.结果:N-2-羟丙基三甲基氯化铵壳聚糖能够在酸性、中性及碱性条件下溶解,并能够与质粒DNA很好的结合形成复合物,拓展了其使用范围.在酸性条件下,N-2-羟丙基三甲基氯化铵壳聚糖与质粒的结合作用强于壳聚糖.壳聚糖及N-2-羟丙基三甲基氯化铵壳聚糖在酸性条件下,30 min即可与质粒形成稳定的复合物,并在12 h内保持良好的稳定性.Hela细胞的体外转染结果表明,N-2-羟丙基三甲基氯化铵壳聚糖的转染效率大于壳聚糖.结论:N-2-羟丙基三甲基氯化铵壳聚糖所适用的pH值范围较壳聚糖更加广泛,其稳定性与壳聚糖无明显差别,并且对Hela细胞的体外转染效率略高于壳聚糖,值得对其进行更深入的研究.

Transfection efficiency of quaternary chitosan/DNA complex in Hela cells

BACKGROUND: Chitosan is one of the most significant non-viral vector materials with the advantages of outstanding biocompatibility. Quarternary chitosan derivatives can improve transfection efficiency and solubility of chitosan in a broader range of pH values. OBJECTIVE: To synthesize a new vector of quarternary chitosan and to study its complex conditions with plasmid and transfection efficiency compared with chitosan. DESIGN, TIME AND SETTING: A contrast observational study was performed in Second Affiliated Hospital of School of Medicine of Zhejiang University between August and October 2008. MATERIALS: Quarternary chitosan was synthesized in Polymer Materials Lab of Beijing Institute of Technology. Plasmid pEGFP-C1 was presented friendly by Mr. Zheng of School of Medicine of Zhejiang University. Hela cells were provided by Miss. Cheng of School of Medicine of Zhejiang University. METHODS: Quarternary chitosan was prepared according to mass concentration of 0.2 g/L, pH value 5.5 (or 6.9, 7.6) and sodium acetate concentration of 50 mmol/L, and rapidly mixed with pEGFP-C1. The mixture was swirled for 15-30 second and stood at room temperature for 30 minutes at least. MAIN OUTCOME MEASURES: The impacts of pH values and time on complex ability of quarternary chitosan and plasmid were studied by gel retardation test. Transfection efficiency of quarternary chitosan on Hela cells was observed by inversed fluorescence microscope and also compared with chitosan. RESULTS: Quarternaty chitosan could form complex with plasmid in acidic, neutral and basic conditions. It could be used in a broader range of pH values. In an acidic condition, the combination of quarternary chitosan with plasmid was superior to chitosan. A stable complex was formed via a combination of quarternary chitosan or chitosan with plasmid within 30 minutes, and the stability lasted for 12 hours. Transfection efficiency of quarternary chitosan on Hela cells demonstrated that transfection efficiency of quarternary chitosan was superior to chitosan. CONCLUSION: Quarternary chitosan has a broader range in use and higher transfection efficiency than chitosan; however, there is no significant difference in stability between quarternary chitosan and chitosan. Additionally, transfection efficiency of quarternary chitosan on Hela cells is superior to chitosan, which needs a further research.