柠檬酸铁铵对悬浮HEK293细胞转染的影响机制探究  

Mechanism Investigation of Ferric Ammonium Citrate on Transfection for Suspended HEK293 Cells

作　　者：陈中元 王玉红 代为俊 张艳敏 叶倩 刘旭平 谭文松[1] 赵亮 CHEN Zhong-yuan;WANG Yu-hong;DAI Wei-jun;ZHANG Yan-min;YE Qian;LIU Xu-ping;TAN Wen-Song;ZHAO Liang(East China University of Science and Technology,Shanghai 200237;Yebio Bioengineering Co.,Ltd.of Qingdao,Qingdao 266114;Shanghai Bioengine Sci-Tech Co.,Ltd.,Shanghai 201203)

机构地区：[1]华东理工大学生物工程学院,上海200237 [2]青岛易邦生物工程有限公司,青岛266114 [3]上海倍谙基生物科技有限公司,上海201203

出　　处：《生物技术通报》2023年第9期311-318,共8页Biotechnology Bulletin

摘　　要：基于聚乙烯亚胺(polyethyleneimine,PEI)的悬浮人胚胎肾细胞(human embryonic kidney 293 cells,HEK293)转染技术前景广阔,但培养基组分会影响转染效率。简单的离心换液存在污染风险,且影响细胞状态。探究抑制转染效率的关键组分对转染过程的影响机制,有利于从根本上解决该问题。首先通过探究培养基中对转染效率具有潜在影响的组分确定关键组分柠檬酸铁铵(ferric ammonium citrate,FAC)的抑制作用,然后通过考察柠檬酸铁铵添加对细胞状态、PEI与DNA形成的复合物(PEI-DNA complex,PEI-DNA复合物)结合情况、目的基因表达情况的影响,分析其抑制转染效率的机制。结果表明,高浓度的柠檬酸铁铵对细胞转染存在明显抑制作用,且该抑制作用随着柠檬酸铁铵浓度的升高而逐渐增强。当柠檬酸根和铁离子同时存在时才会抑制细胞转染过程。过高浓度的柠檬酸铁铵使PEI-DNA复合物的粒径显著增加,复合物进入细胞更加困难,导致进入细胞的复合物数量减少,最终引起细胞转染效率的大幅下降。柠檬酸铁铵通过影响PEI-DNA复合物的大小限制DNA进入细胞,从而抑制PEI介导的悬浮HEK293细胞转染过程。控制转染时柠檬酸铁铵浓度在20μmol/L内可解决其对转染过程的抑制作用。本研究深入认识了柠檬酸铁铵对PEI介导的悬浮HEK293细胞转染过程的影响及其机制,为悬浮HEK293细胞转染培养基的开发和理性设计提供有力参考。Suspended HEK293(human embryonic kidney)cell transfection based on PEI(polyethyleneimine)is a prospective technology.However,the components of the medium often affect the transfection efficiency,which needs to be changed with transfection medium by centrifugation.This process is cumbersome and not only has the risk of contamination,but also affects the cell state.In order to fundamentally solve this problem,the influence mechanism of key components that inhibit the efficiency of transfection on the transition process need to be explored.First we explored that the key composition that have potential effects on transfection efficiency in the medium is ferric ammonium citrate(FAC),and determined the its inhibitory effect.Then we analyzed its mechanism for inhibiting transit efficiency,including the effects of ferric ammonium citrate on the cell state,PEI-DNA complex formation,and destination gene expression.Results showed that certain amount of FAC had significant inhibitory effect on the transfection that was also gradually enhanced with concentration increasing.Further studies found that the transfection was inhibited only when citrate and iron ions were present at the same time.Over high concentration of FAC increased the particle size of the PEI-DNA complex,making it more difficult to enter the cell.This led to a reduction in the number of complexes entering the cells,and ultimately resulted in a significant drop in cell transfection efficiency.FAC restricted DNA into cells by affecting the size of the PEIDNA complex,thereby inhibiting PEI-mediated suspended HEK293 cell transfusion process.The inhibitory effect can be eliminated when the FAC concentration was<20μmol/L.Through this study,the effect of FAC on the transfection process of PEI-mediated suspended HEK293 cells and its mechanism were deeply understood,providing a solid reference for the development and rational design of the transfection medium for suspended HEK293 cells.

关 键 词：悬浮HEK293细胞 转染效率 柠檬酸铁铵 PEI介导的转染 哺乳动物细胞瞬时表达系统 

分 类 号：Q813.11[生物学—生物工程]