单端羧基聚乙二醇/鱼精蛋白系统介导HSV-TK基因在宫颈癌细胞中的体外研究  

Study on carboxyl-PEG /protamine-mediated HSV-TK gene transfection in cervical cancer cells

作　　者：谢日志 胡海梅[1] 李晓琳[1] 

机构地区：[1]广东药学院生命科学与生物制药学院,广东广州510006

出　　处：《广东药学院学报》2014年第1期85-91,共7页Academic Journal of Guangdong College of Pharmacy

基　　金：广东省大学生创新实验项目(1057310032)

摘　　要：目的研究非病毒载体MPEG-C-鱼精蛋白系统的制备方法、其对不同剂量鱼精蛋白与单纯疱疹病毒胸苷激酶(HSV-TK)的结合能力以及对HeLa细胞的影响。方法将一定量的MPEG-C-和不同量的鱼精蛋白混合后与DNA室温孵育,得到MPEG-C-鱼精蛋白/DNA复合物;用琼脂糖电泳实验测定不同N/P比形成复合物时对HSV-TK的阻滞情况;用结合沉淀试验比较不同量鱼精蛋白对包裹HSV-TK的能力的影响;MTT法检测MPEG-C-鱼精蛋白对HeLa宫颈癌细胞的毒性作用及其复合物对HeLa细胞的转染率。结果 MPEG-C-鱼精蛋白复合物包裹HSV-TK的能力随N/P比增大而增强;粒径随N/P比增大而减小,可达200 nm左右;复合物的Zeta电位随N/P比增大而增强;与单独鱼精蛋白复合物相比,细胞转染率有所降低。结论 MPEG-C-鱼精蛋白是一种制备工艺简单、对HSV-TK包裹能力高、细胞毒性小、具有一定价值的非病毒载体。Objective To study the preparation of MPEG-C-protamine non-viral vector system, the capability of different dose of protamine binding to HSV-TK, and its influence on HeLa cells. Methods MPEG-C- and different amounts of protamine were mixed, and then MPEG-C-protamine/HSV-TK complexes were prepared after incubation at room temperature. Agarose electrophoresis was used to determine the MPEG-C- protamine complexes binding to HSV-TK with different N/P ratios. Binding precipitation experiment was used to compare the package ability of different amounts of MPEG-C-protamine on HSV-TK. MTY assay was used to evaluate the cytotoxicity of MPEG-C-protamine/HSV-TK complexes on HeLa cells. Transfection efficiency of the complexes on HeLa cells was evaluated. Results When the N/P ratio was increased, the package ability of MPEG-C-protamine complexes on HSV-TK was enhanced, the particle size was reduced to about 200 nm, and Zeta potential of complexes was increased. Compared with the single protamine, transfection rate was decreased in MPEG-C-protamine complexes. Conclusion MPEG-C- protamine is a valuable non-viral vector system, owing to its simple preparation process, strong HSV-TK coating ability and low cytotoxicity.

关 键 词：非病毒基因载体 端羧基聚乙二醇 鱼精蛋白 HSV-TK 基因治疗 体外 

分 类 号：Q782[生物学—分子生物学]