超声靶向微泡联合肽核酸结合核定位信号肽增强基因转染的实验研究  

Experiment study on the transfection of exogenous genes promoted by ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal

作　　者：姜楠[1] 陈茜[1] 胡波[1] 周青[1] 曹省[1] 冯闯丽 郭瑞强[1] 

机构地区：[1]武汉大学人民医院超声影像科,430060

出　　处：《中华超声影像学杂志》2017年第5期442-447,共6页Chinese Journal of Ultrasonography

基　　金：国家自然科学基金青年基金项目（81201109,81501495）

摘　　要：目的应用超声靶向微泡破坏（UTMD）介导连接有核定位信号（NLS）的质粒DNA转染293T细胞，增加基因入胞及人核量，从而提高细胞对外源基因的转染效率。方法合成连接有细胞特异性抗体的靶向微泡，特异性识别293T细胞膜表面SV40T抗原，强化UTMD空化作用使细胞膜通透性增加，从而提高质粒入胞率；通过肽核酸（PNA）将NLS与增强型绿色荧光蛋白质粒（EGFP—N3）基因交联，构建一种基因传递系统介导细胞核对外源基因的摄取。转染后比较以下几组间转染效率：空白对照组（A组）、普通微泡＋质粒组（B组）、靶向微泡＋质粒组（C组）、普通微泡＋NLS-PNA-DNA组（D组），靶向微泡＋NLS-PNA-DNA组（E组）。倒置荧光显微镜观察荧光分布情况；CCK-8检测细胞活性；流式细胞仪检测细胞转染率；RT-PCR以及Western blot检测目的基因mRNA和蛋白的表达水平。结果UTMD联合靶向微泡可显著提高胞质对外源性DNA的摄入，且细胞存活率均〉80％；倒置荧光显微镜示细胞内绿色荧光表达量依次递增；流式细胞仪检测五组基因转染效率分别为0、（9．30±0．46）％、（26．46±2．01）％、（29．54±0．62）％、（45．72±1．86）％，差异均有统计学意义（P均〈0．05）；E组EGFP的mRNA和蛋白的相对表达量均显著高于C、D组，差异均有统计学意义（P均d0．05）。结论UTMD介导靶向微泡和连接有核定位信号的质粒DNA可明显增加细胞对质粒DNA的摄取表达，显著提高外源基因的转染效率。Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery（UTMD） mediated a peptide nucleic acid （PNA） binding nuclear localization signal （NLS）. Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor. The SV40T antigen receptors were expressed on the membranes of 293T cells. The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA, which increased nuclear localization. Ultrasound-targeted microbubble delivery （UTMD） and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm, respectively. The study was divided into five groups：Contrast （group A）, Common microbubble ＋ DNA （group B）, Antibody-targeted microbubbles ＋ DNA （group C）, Common microbubbles ＋ NLS-PNA-DNA （group D）, Antibody-targeted microbubbles ＋ NLS-PNA-DNA （group E）. Fluorescence microscope was used to observe the fluorescent light in each group;flow cytometry to test the transfection;RT-PCR and Western blot to detect genesr mRNA and protein expression level. Results Ultrasound and antibody-targeted microbubble delivery （UTMD） significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability（〉80%）. Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully. The transfection results of flow cytometry were 0, （9.30 ± 0.46）%, （26.46 ±2.01）%, （29.54 ± 0.62） %, （45.72 ± 1.86） %, respectively,and the differences were statistically significant（ P〈0.05）. The relative mRNA and protein expression in group E were greater than those in group C and D respectively （ P 〈0.05）.Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nucl

关 键 词：超声处理 微气泡 肽核酸 核定位信号 转染 

分 类 号：R450[医药卫生—治疗学]