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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。
Aim To investigate the effect of the polar charge domain of the penetrating peptide AR-23 [melittin-related peptide from Rana tagoi] on cell membrane penetration and its role in promoting polyethylenimine (PEI) -mediated The role of gene transfection. Methods The transmembrane peptide AR-23 and the hydrophobic core region AR-20 were synthesized. The secondary structure was analyzed by circular dichroism (CD). The cell membrane penetrating ability was compared by hemolysis assay. Polarization experiments were carried out to analyze the interaction of membrane penetrating peptide and cell membrane. The penetrating peptide and PEI were mixed in a certain proportion. The detection genes of GFP and luciferase were detected in HeLa and RAW264.7 Transfection efficiency in cells was measured by MTT assay of cell toxicity after gene transfection. Results AR-23 showed a typical α-helical structure in the simulated cell membrane environment with methanol, the α-helix rate was 37.6%, while AR-20 did not show the typical α-helical structure; AR-23 can penetrate the cell membrane to cause hemolysis of erythrocytes while AR-20 does not have hemolytic effect; AR-23 has strong depolarization to cell membrane, while AR- AR-23 could obviously enhance the transfection efficiency of PEI-mediated gene in HeLa and RAW264.7 cells, and did not cause the S toxicity of transfected cells. AR-20 could not enhance the efficiency of gene transfection. Conclusion The polar charge region of AR-23 is crucial for maintaining its α-helical structure and the ability to penetrate the membrane. AR-23 can enhance PEI-mediated gene transfection efficiency without causing cytotoxicity and is an ideal enhancer for gene transfection.