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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。
Objective To study the mechanism of transmembrane peptide enhancing gene transfection efficiency of branched polyethyleneimine (BPEI) in CHO-K1 cells. Methods The melittin and its hydrophobic core region MT20 were synthesized and their secondary structures were analyzed by circular dichroism spectroscopy (CD). The ability of penetrating cell membranes was compared by hemolysis test. Before and after addition of melittin and MT20, To observe the distribution of calcein in HeLa cells. The melittin or MT20 was mixed with BPEI in a certain proportion respectively. The luciferase reporter gene was used to detect the transfection efficiency of luciferase in CHO-K1 cells. The transfected cells were detected by MTT assay toxicity. Results Both melittin and MT20 showed a certain α-helical structure in the methanol solution simulating membrane environment with the proportions of 59.63% and 35.67%, respectively, indicating that their secondary structure was related to the transmembrane function; Penetrating the cell membrane under neutral conditions lead to hemolysis of erythrocytes while MT20 has the ability to penetrate the membrane under both neutral and acidic conditions; addition of melittin and MT20 can promote the distribution of calcein in the MT20 group, The results showed that both MT20 and melittin could enhance the transfection efficiency of BPEI in CHO-K1 cells, the enhancement effect of MT20 was stronger and the cytotoxicity was stronger than that of melittin and single Lower when using BPEI and Lipofectamine 2000. Conclusion MT20, a hydrophobic core of melittin, is a low toxicity gene transfection enhancer by increasing the escape of PEI / DNA complex particles from inclusions and promoting their transfection into the nucleus. It plays an important role in difficult-to-transfect cell lines.