多聚胺阳离子脂质体转染体系的构建

Construction of polyamine cationic liposome transfection system

目的制备多聚胺阳离子脂质体(PolyamineCationicliposome,PCL),转染哺乳动物细胞,评估其转染效率及细胞毒性,筛选高效低毒的阳离子脂质体转染体系。方法多聚胺阳离子脂质TC-Chol与中性磷脂DOPE以一定比例,制备PCL,通过转染以增强型绿色荧光蛋白为报告基因的质粒PIRES2-EGFP入Hela细胞,倒置荧光显微镜下检测转染细胞的报告基因表达,通过MTT法,检测细胞毒性,筛选阳离子脂质体与DNA结合的最佳比例及最佳转染时间。结果多聚胺阳离子脂质体与DNA的质量比为3～4∶1时可达到高效低毒,转染后48h转染效率最高。结论由TC-Chol与DOPE制备的多聚胺阳离子脂质体可提高转染效率,在一定条件下可达到相对高效低毒,为基因转染和基因治疗提供可靠的实验室依据。

[Objective] To prepare polyamine cationic liposome（PCL）, and transfect it into mammlian cell to assess transfection efficiency and cytotoxicity, and to optimize the condition of transfection. [Methods] PCL with polyamine cationic lipids TC-Chol and neutral phospholipid DOPE at a certain molar ratio was prepared, the plasmid PIRES2-EGFP which mediated PCL was transfected into Hela cells, and the expression of EGFP was determined by fluorescence microscope. The survival fraction rate was determined by MTT to evaluate the cytotoxicity post-transfection for optimization of the transfection condition of mammalian cell. [Results] PCL mediated transfection could enhance the transfection efficiency, at the mass ratio of 3-4：1 and it was more efficient and less cytotoxic. [Conclusion] PCL prepared with TC-Chol and DOPE can enhance the transfection efficiency, and satisfactory transfection activity and less cytotoxicity is observed under certain condition. It provides laboratary data for gene transfection and gene therapy.