穿膜肽AR-23增强聚乙烯亚胺介导的基因转染效率及其机制的初步研究  

Effect of positively charged residues at C-terminal of AR-23 on improvement of polyethylenimine-mediated gene transfection

作　　者：马倩[1] 檀英霞[2] 王颖丽[2] 李素波[2] 高红伟[2] 鲍国强[2] 季守平[2] 

机构地区：[1]中南大学生物科学与技术学院,长沙410012 [2]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学》2013年第5期349-353,共5页Military Medical Sciences

摘　　要：目的研究穿膜肽AR-23[蜂毒肽相关肽,来自日本蛙(Rana tagoi)]的极性电荷区对细胞穿膜作用的影响及其在促进聚乙烯亚胺(polyethylenimine,PEI)介导的基因转染中的作用。方法分别合成了穿膜肽AR-23及由疏水核心区组成的截短肽AR-20;利用圆二色谱(CD)分析二级结构;通过溶血实验比较两者的细胞膜穿透能力;利用去极化实验分析穿膜肽与细胞膜的相互作用方式;将穿膜肽与PEI按一定比例混合,以绿色荧光蛋白(GFP)和荧光素酶(luciferase)为报告基因检测基因在HeLa和RAW264.7细胞中的转染效率,用MTT法测定基因转染后细胞的毒性。结果在以甲醇模拟的细胞膜环境中,AR-23呈现出典型的α-螺旋结构,α-螺旋率为37.6%,而AR-20未呈现出典型的α-螺旋结构峰;在偏酸性和中性条件下,AR-23均能穿透细胞膜导致红细胞溶血,而AR-20不具备溶血作用;AR-23对细胞膜具有较强的去极化作用,而AR-20去极化能力极弱;AR-23可明显增强PEI介导的基因在HeLa和RAW264.7细胞中的转染效率,并且不引起转染后细胞的S毒性,AR-20则不具备增强基因转染效率的作用。结论 AR-23的极性电荷区对于维持其α-螺旋结构和穿膜能力至关重要。AR-23能够提高PEI介导的基因转染效率而不会导致细胞毒性,是一种理想的基因转染增强剂。）Objective To investigate the effect of positively charged residues of AR-23 [a melittin-related peptide（MRP） from Rana tagoi] on membrane penetrating activity and polyethylenimine（PEI）-mediated gene transfection.Methods AR-23 and its truncated analog AR-20（without positively charged residues of AR-23） were synthesized.Then,the second structural information and penetrating ability of peptides were examined by CD spectroscopy and hemolytic activity,respectively.The interaction of peptides with membrane was detected by depolarization study.The effect of peptides on PEI-mediated gene transfection was evaluated using green fluorescence protein（GFP） and luciferase as reporter genes.The viability of transfected cells was evaluated by MTT assay.Results In the mimetic membrane environment,AR-23 showed typical α-helical structure in which the α-helical content was 37.6%,but AR-20 did not present α-helical conformation.AR-23 exhibited concentration-dependent hemolysis under both neutral and acidic conditions,but AR-20 did not have hemolytic activity.Compared to AR-20,cell membrane was efficiently depolarized by AR-23.Trans fection results showed that luciferase activity and GFP expression in AR-23 /PEI/DNA transfected cells were higher than in PEI/DNA group in both HeLa and RAW264.7 cells.Compared with PEI transfected group,AR-23 group had higher cell viability in HeLa and RAW264.7cells.Conclusion The positively charged residues at C-terminal of AR-23 play an important role in maintaining α-helical conformation and membrane penetrating activity.Furthermore,PEI mediated transfection can be effectively improved by AR-23 without causing significant cytotoxicity.Therefore,AR-23 may be considered a potential enhancer of cationic polymers mediated transfection efficiency.

关 键 词：蜂毒肽 穿膜肽 聚乙烯亚胺 膜穿透活性 基因转染 

分 类 号：R392[医药卫生—免疫学]