酸敏感阳离子聚合物基因载体bPOEAA的体外转染条件优化
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摘要
目的探讨不同体外转染条件对酸敏感阳离子聚合物基因载体bPOEAA包裹的荧光素酶质粒pGL3复合物转染效率的影响。方法获得最佳转染效率后,利用bPOEAA包裹绿色荧光蛋白质粒pEGFP-N1,转染非洲绿猴肾成纤维细胞COS-7和小鼠成纤维细胞NIH3T3,利用荧光显微镜和流式细胞术考察转染效率。结果最佳转染条件确定为:COS-7细胞在pH值为7.4、复合物中所含DNA量为5μg、bPOEAA/pGL3质量比为24转染时使用含有10%血清的DMEM培养基。优化后荧光素酶质粒表达量为优化前的31倍,细胞转染效率达38.6%。结论培养基pH值、DNA量、bPOEAA/pGL3质量比、血清含量对复合物转染效率均有显著影响。
Objective To investigate the effect of different transfection conditions on transfection efficiency of luciferase plasmid pGL3 and acid-sensitive cationic polymer gene carrier bPOEAA complexes in vitro.Methods A green fluorescence protein reporter gene pEGFP-N1 was added to bPOEAA to prepare polyplex for transfection to African green monkey kidney fibroblast cells COS-7 and mouse fibroblast cells NIH3T3 at the optimal condition in vitro.FACSCalibur flow cytometer and fluorescence microscopy were used to analyze transfection efficiency.Results The optimal transfection conditions were pH value of culture medium as 7.4,5μg of plasmid DNA contained in polyplex and the weight ratio of bPOEAA to pGL3 as 24 in COS-7 cells with DMEM medium containing 10% serum.After optimization,the expression of luciferase plasmid was 31 times as much as that before,and the transfection efficiency was 38.6%.Conclusion The trasnfection efficiency was greatly affected by pH value of culture medium,the amount of plasmid DNA,the weight ratio of bPOEAA to pGL3 and serum content.
Objective To investigate the effect of different transfection conditions on transfection efficiency of luciferase plasmid pGL3 and acid-sensitive cationic polymer gene carrier bPOEAA complexes in vitro.Methods A green fluorescence protein reporter gene pEGFP-N1 was added to bPOEAA to prepare polyplex for transfection to African green monkey kidney fibroblast cells COS-7 and mouse fibroblast cells NIH3T3 at the optimal condition in vitro.FACSCalibur flow cytometer and fluorescence microscopy were used to analyze transfection efficiency.Results The optimal transfection conditions were pH value of culture medium as 7.4,5μg of plasmid DNA contained in polyplex and the weight ratio of bPOEAA to pGL3 as 24 in COS-7 cells with DMEM medium containing 10% serum.After optimization,the expression of luciferase plasmid was 31 times as much as that before,and the transfection efficiency was 38.6%.Conclusion The trasnfection efficiency was greatly affected by pH value of culture medium,the amount of plasmid DNA,the weight ratio of bPOEAA to pGL3 and serum content.
引文
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[2]Liu WM,Liu M,Xue YN,et al.Poly(amidoamine)s with pendant primary amines and flexible backbone for enhanced nonviral gene delivery:transfection and intracellular trafficking[J].J Biomed Mater Res A,2012,100A(4):872-881.
[3]Gabrielson NP,Lu H,Yin L,et al.Reactive and bioactive cationicα-helical polypeptide template for nonviral gene delivery[J].Angew Chem Int Ed Engl,2012,51(5):1143-1147.
[4]Wang H,Wang Y,Wang Y,et al.Self-assembled fluorodendrimers combine the features of lipid and polymeric vectors in gene delivery[J].Angew Chem Int Ed Engl,2015,54(40):11647-11651.
[5]Favretto ME,Krieg A,Schubert S,et al.Multifunctional poly(methacrylate)polyplex libraries:aplatform for gene delivery inspired by nature[J].J Control Release,2015,209:1-11.
[6]Buschmann MD,Merzouki A,Lavertu M,et al.Chitosans for delivery of nucleic acids[J].Adv Drug Deliv Rev,2013,65(9):1234-1270.
[7]Xu FJ,Yang WT.Polymer vectors via controlled/living radical polymerization for gene delivery[J].Prog Polym Sci,2011,36(9):1099-1131.
[8]Medina-kauwe LK,Xie J,Hamm-alvarez S.Intracellular trafficking of nonviral vectors[J].Gene Ther,2005,12(24):1734-1751.
[9]Schaffer DV,Fidelman NA,Dan N,et al.Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery[J].Biotechnol Bioeng,2000,67(5):598-606.
[10]Wang R,Cheng J,Yan G,et al.Rational design of acid-labile branched polycation with superior gene transfection capacity[J].Polymer,2017,123:1-9.
[11]Sato T,Ishii T,Okahata Y.In vitro gene delivery mediated by chitosan.Effect of pH,serum,and molecular mass of chitosan on the transfection efficiency[J].Biomaterials,2011,22(15):2075-2080.
[12]Goldman CK,Soroceanu L,Smith N,et al.In vitro and in vivo gene delivery mediated by a synthetic polycationic amino polymer[J].Nat Biotechnol,1997,15(5):462-466.
[13]陶扬洋,巩凯,王睿,等.新型聚原酸酯共聚物的合成及性能研究[J].高分子通报,2013(6):33-38.