组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2019, Vol. 15 ›› Issue (6): 373-383.doi: 10.3969/j.issn.1673-0364.2019.06.001

• 论著 •    下一篇

脂质体方法用于modRNA 转染各种类型细胞的初步研究

颜冰倩，王会景，艾雪峰，宫艺其，谭瑶，徐徐，王伟，付炜   

  1. 上海交通大学医学院附属上海儿童医学中心转化医学研究所，胸心外科；上海市组织工程重点实验室
  • 收稿日期:2019-11-10 修回日期:2019-12-06 出版日期:2019-12-20 发布日期:2019-12-20
  • 作者简介:付炜(E-mail:fuweizhulu@163.com)。

Preliminary Research of Liposome in modRNA Transfection to Different Cell Types

YAN Bingqian,WANG Huijing,AI Xuefeng,GONG Yiqi,TAN Yao,XU Xu,WANG Wei,FU Wei   

  1. Institute of Pediatric Translational Medicine, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine; Department of Pediatric Cardiothoracic Surgery, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine;Shanghai Key Laboratory of Tissue Engineering
  • Received:2019-11-10 Revised:2019-12-06 Online:2019-12-20 Published:2019-12-20
  • Contact: 国家自然科学基金项目(81670372)；上海市浦江人才计划(18PJD031)；上海市转化医学协同创新中心项目(TM201821)；上海交通大学医工交叉项目(YG2016MS28)；上海交通大学医学院紧缺专业硕士研究生临床研究能力提升计划(EKKY2018002DGD)；上海市组织工程重点实验室开放课题

摘要： 目的筛选更优的脂质体转染试剂,探讨其用于modRNA转染各种类型细胞并表达所需目的蛋白的可行性。方法分别用RNAiMax及MessageMax将modGFP转染入MEF细胞,应用荧光显微镜、流式细胞分析仪检测并比较两种转染试剂的转染效率和蛋白表达效果。将更优的转染试剂用于modGFP转染其他各种类型细胞的实验中,采用同样的方法分析modGFP的转染效率及蛋白表达情况。将modGFP与modmCherry共转MEF细胞,荧光显微镜观察共转的情况。将modnGFP及modmTBX5转染MEF细胞,荧光显微镜观察核内蛋白的表达情况。结果 MEF细胞转染modGFP 24 h后,流式分析发现MessageMax的转染效率为(83.33%±3.23%),略高于RNAiMax的(78.77%±6.12%),两者没有统计学差异,但是流式分析和荧光显微镜观察均表明MessageMax转染后1周内的蛋白翻译表达效率更高,是更高效的modRNA转染试剂。MessageMax能将modC;FP高效转染人3T3细胞、Hela细胞及MCF-7细胞中,并实现modGFP和modmCherry在MEF细胞中的共转及核内因子nC;FP和mTBX5在MEF细胞核中的定位。结论相比于RNAiMax,MessageMax转染modRNA效果更佳,既能实现包括核内蛋白在内的各种目的蛋白在各类细胞中的高效、快速表达,也能实现多因子同时转染、共同表达。

关键词: 化学合成修饰信使核糖核酸, 脂质体, 基因表达, 小鼠胚胎成纤维细胞

Abstract: Objective To screen a better liposome agent for modRNA transfection and to explore the feasibility and efficiency of using it to transfect various cell types and express target proteins. Methods After modGFP was transfected into MEF cells with RNAiMax and MessageMax, the transfection efficiency and protein expression effects of the two transfection agents were detected and compared by fluorescence microscope and flow cytometry. The better liposome agent was chosen for modGFP transfection into various cell types, and the transfection and expression efficiency were measured using the same methods. Besides, the co-transfection of modGFP and modmCherry, and the nuclear transfection of modnGFP and modmTBX5 into MEF cells were observed by fluorescence microscope. Results After MEF cells were transfected with modGFP for 24 h, flow analysis showed that the transfection efficiency of MessageMax(83.33%±3.23%) was slightly higher than that of RNAi Max(78.77%±6.12%), but there was no statistical difference. However, both flow analysis and fluorescent images found that MessageMax had a higher efficiency of protein expression within 1 week after transfection, making it a better modRNA transfection reagent. MessageMax could efficiently transfect modGFP into 3T3 cells, Hela cells and MCF-7 cells, achieve co-translocation of modGFP and modmCherry, and achieve nuclear localization of intranuclear factors nGFP and mTBX5 in MEF cells. Conclusion Compared to RNAi Max, modRNA transfection with MessageMax brings better results, which can not only achieve efficient and rapid expression of various target proteins including nuclear proteins in various cell types, but also realize simultaneous transfection and expression of multiple factors.

Key words: Chemically synthetic modified message RNA, Liposome, Gene expression, Mouse embryonic fibroblast

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