Role of specific endocytic pathways in electrotransfection of cells.
Role of specific endocytic pathways in electrotransfection of cells.
复制标题
DOI：
10.1038/mtm.2014.58
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
电转染是一种在临床前和临床研究中用于基因传递的技术。然而，其机制尚不完全清楚。本研究的目的是研究电转染中涉及的内吞作用的特定途径。在该研究中，三种不同的人类细胞系（HEK293、HCT116 和 HT29）在电转染后用冰冷培养基处理，或在电转染前用内吞抑制剂处理。这些抑制剂是药物（氯丙嗪、染料木黄酮和阿米洛利）或不同的小干扰RNA（siRNA）分子，它们分别可以敲低网格蛋白重链（CLTC）、caveolin-1和Rab34的表达。 siRNA 处理后 48-72 小时，通过蛋白质印迹分析证实了基因表达的减少。据观察，与匹配的对照相比，用冰冷培养基、氯丙嗪或染料木黄酮处理会导致所有三种细胞系的电转染效率（eTE）显着降低，但阿米洛利处理对 eTE 的影响不显着。对于用 siRNA 处理的细胞，只有 CLTC 敲低导致所有三种细胞系的 eTE 减少。总之，这些数据表明网格蛋白介导的内吞作用在电转染中发挥重要作用。
Electrotransfection is a technique utilized for gene delivery in both preclinical and clinical studies. However, its mechanisms are not fully understood. The goal of this study was to investigate specific pathways of endocytosis involved in electrotransfection. In the study, three different human cell lines (HEK293, HCT116, and HT29) were either treated with ice cold medium postelectrotransfection or endocytic inhibitors prior to electrotransfection. The inhibitors were pharmacological agents (chlorpromazine, genistein, and amiloride) or different small interfering RNA (siRNA) molecules that could knockdown expression of clathrin heavy chain (CLTC), caveolin-1, and Rab34, respectively. The reduction in gene expressions was confirmed with western blot analysis at 48-72h post-siRNA treatment. It was observed that treatments with either ice cold medium, chlorpromazine, or genistein resulted in significant reductions in electrotransfection efficiency (eTE) in all three cell lines, compared to the matched controls, but amiloride treatment had insignificant effects on eTE. For cells treated with siRNA, only CLTC knockdown resulted in eTE reduction for all three cell lines. Together, these data demonstrated that the clathrin-mediated endocytosis played an important role in electrotransfection.
登录
查看更多内容
DOI：
10.1083/jcb.200302028
发表时间：
2003-05-26
期刊：
The Journal of cell biology
影响因子：
--
作者：
Nabi IR;Le PU
通讯作者：
Le PU
DOI：
10.1083/jcb.131.1.69
发表时间：
1995-10
期刊：
The Journal of cell biology
影响因子：
--
作者：
Damke H;Baba T;van der Bliek AM;Schmid SL
通讯作者：
Schmid SL
影响因子：
3.3
作者：
Amyere, M;Payrastre, B;Courtoy, PJ
通讯作者：
Courtoy, PJ
影响因子：
5.8
作者：
ELFERINK, JGR
通讯作者：
ELFERINK, JGR
影响因子：
3.5
作者：
Faurie, Cecile;Rebersek, Matej;Rols, Marie-Pierre
通讯作者：
Rols, Marie-Pierre