聚乙烯亚胺转基因影响因素的测定及其优化  

Identification and Optimization of Influencing Factors on Polyethylenimine Transfection Efficiency

作　　者：李经忠[1] 王青青[1] 余海[1] 沈芬平[1] 吴萍萍[1] 

机构地区：[1]浙江大学免疫学研究所,杭州310031

出　　处：《中国生物化学与分子生物学报》2004年第2期234-240,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家高技术研究发展计划资助 (863 ) (编号 :2 0 0 1AA2 170 71)~~

摘　　要：聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .Polyethylenimine(PEI) polycation can condense DNA into granules of around nanometer size. which can be used for delivering gene into mammalian cells. This paper studied the influencing factors on polyethylenimine ( M r 25?000, branched) gene delivery efficiency in order to develop a more complicated nonviral gene delivery vector. \%In vitro\% cytotoxic effects of polyethylenimine were quantified by MTT assay.When polyethylenimine incubated with the cells for 24 hours, the survival rate of COS\|7 cells was 80% with 6 μg/ml of polyethylenimine,64% with 7 μg/ml.40% with 8 μg/ml. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3 0 polyethylenimine nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of polyethylenimine. Bovine serum albumin and bovine serum in the culture medium decreased the transfection efficiency. The transfection efficiency of PEI/DNA diluted with 0 9% NaCl was higher than that with 5% glucose. After adding PEI/DNA complex, level shake (≤30 min) of culture plates improved the transfection efficiency. Polyethylenimine was an efficient gene transfection agent of eukaryotes in vitro, and could be used for synthesizing more complicated gene delivery carriers for in vivo gene therapy.

关 键 词：聚乙烯亚胺 转基因 基因治疗 基因释放载体 

分 类 号：Q78[生物学—分子生物学]