穿膜肽增强聚乙烯亚胺介导的基因转染效率的机制研究  

Enhancing gene transfection efficiency of polyethylenimine by hydrophobic core of melittin

作　　者：陈翀[1] 檀英霞[1] 王颖丽[1] 李素波[1] 季守平[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学》2011年第6期442-446,共5页Military Medical Sciences

摘　　要：目的研究穿膜肽增强支链聚乙烯亚胺(BPEI)在CHO-K1细胞中的基因转染效率的机制。方法分别合成蜂毒肽(melittin)及其疏水核心区MT20,利用圆二色光谱(CD)分析其二级结构;通过溶血试验比较二者穿透细胞膜的能力;加入蜂毒肽和MT20前后,观察钙黄绿素(calcein)在HeLa细胞内的分布情况。分别将蜂毒肽或MT20与BPEI按一定比例混合,以荧光素酶(luciferase)为报告基因,检测荧光素酶在CHO-K1细胞中的转染效率;以MTT法检测基因转染后的细胞毒性。结果在模拟膜环境的甲醇溶液中,蜂毒肽和MT20都呈现一定的α-螺旋结构,比例分别为59.63%和35.67%,说明其二级结构与之穿膜功能相关;蜂毒肽只能在中性条件下穿透细胞膜导致红细胞溶血,而MT20在中性和酸性条件下都具有穿膜能力;加入蜂毒肽和MT20后能够促进钙黄绿素在MT20组中呈弥散分布,在蜂毒肽组中呈颗粒状分布并且在核仁内蓄积;MT20和蜂毒肽都能够增强BPEI在CHO-K1细胞中的基因转染效率,其中MT20的增强作用更强且细胞毒性较蜂毒肽以及单独使用BPEI和Lipofectamine 2000时要低。结论蜂毒肽的疏水核心区MT20通过增加PEI/DNA复合物颗粒从内含体逃逸并促进其进入细胞核而增加其基因转染效率,是一种低毒的基因转染增强剂,有可能在难以转染的细胞系中发挥重要作用。ObjectiveTo study the role of cell penetration peptide in enhancing branched polyethylenimine（BPEI） mediated gene transfection efficiency in CHO-K1 cells.MethodsMelittin and its hydrophobic core（MT20） were synthesized and their secondary structure was analyzed with circular dichroism spectra scanning;their relative membrane penetration activity was evaluated by detecting the hemolysis level in pH 7.4 or pH 5.4 buffer;the distribution of calcein in HeLa cells before and after melititn/MT20 treatment was observed.PEI and melittin /MT20 were mixed at different ratios before binding to luciferase plasmids and the transfection efficiency was assayed by detecting luciferase activity in CHO-K1 cells after gene transfection.The cytotoxicity was analyzed by MTT assay at 48 hours after transfection.ResultsIn the mimetic membrane environment（methanol solution）,melittin and MT20 presented 59.63% and 35.67% of α-helix,respectively;melittin had hemolysis potency in neutral or basic environments,but its hemolysis potency sharply decreased in an acid environment.However,MT20 maintained the determinate hemolysis potency in an acid environment;after melittin or MT20 treatment,a large amount of calcein was diffused in whole cells in MT20 group and accumulated in nucleus specifically in melittin group.Both melittin and MT20 could enhance gene transfection of BPEI in CHO-K1 cells.When MT20 enhanced group with PEI group and Lipofectamine 2000 group were compovred,the highest transfection efficiency（3.31×109 RLU/mg luciferase protein） and the lowest cell toxicity（88.75% cell viability） were obtained in MT20 enhanced group.ConclusionThe hydrophobic core of melittin（MT20） enables efficient vesicular escape and enhances nuclear access of nonviral gene delivery vectors（BPEI）,consequently enhancing gene transfection efficiency.It is concluded that the hydrophobic core of melittin may be a potential enhancer of PEI that will be used to improve gene transfection in difficult-to-transfect cells.

关 键 词：穿膜肽 聚乙烯亚胺 转染效率 疏水核心区 

分 类 号：R346[医药卫生—基础医学]