电转方法用于modRNA转染细胞的初步研究  

Preliminary Study of Electroporation Method for modRNA Transfected Cells

作　　者：陈茂林 王会景 艾雪峰 颜冰倩 宫艺奇 谭瑶 付炜[2,3] 王伟 CHEN Maolin;WANG Huijing;AI Xuefeng;YAN Bingqian;GONG Yiqi;TAN Yao;FU Wei;WANG Wei(Department of Pediatric Cardiothoracic Surgery;Institute of Pediatric Translational Medicine,Shanghai Children’s Medical Center,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China;Shanghai Key Laboratory of Tissue Engineering,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属上海儿童医学中心心胸外科,上海市200127 [2]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,上海市200127 [3]上海市组织工程研究重点实验室,上海市200011

出　　处：《组织工程与重建外科杂志》2019年第4期215-221,共7页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家自然科学基金项目(81670372);上海市浦江人才计划(18PJD031);上海交通大学医学院转化医学协同创新中心合作研究项目(TM201821);上海交通大学医工交叉项目(YG2016MS28);上海市组织工程重点实验室开放课题

摘　　要：目的探讨电转方法用于modRNA转染细胞的可行性,并筛选出不同细胞电转所需的最适电压和脉冲时间。方法①筛选电转EGFPmodRNA进入人成纤维细胞的最佳电压和脉冲时间参数;②在此基础上将人成纤维细胞转染nGFP/EGFP+mCherrymodRNA;③将Hela、293T、3T3三种常见贴壁细胞电转EGFPmodRNA。结果①人成纤维细胞电转modRNA的最适电压为440V,最适脉冲时间为30ms;②nGFP可在核内表达,EGFP和mCherry两种蛋白可同时在胞质中表达;③Hela、293T、3T3细胞电转最适电压分别为425V、400V和440V,最适脉冲时间均为30ms。结论本实验证明①电转法转染modRNA进入细胞质和细胞核的可行性;②可同时将两种modRNA导入同一个细胞内并表达;③最适电压和最适脉冲时间需根据不同的细胞种类分别筛选。Objective To investigate the feasibility of electroporation for modRNA transfection and to screen the optimal voltage and pulse time required for different cell electroporation. Methods ①The optimal voltage and pulse time parameters for electroporation of EGFP modRNA into human fibroblasts were screened;②Human fibroblasts were transfected with nGFP and EGFP+mCherry modRNA;③Hela, 293T, 3T3, three common adherent cells were electroporated with EGFP modRNA. Results ①The optimum voltage for electroporation of human fibroblasts was 440 V, and the optimal pulse time was 30 ms;②nGFP could be expressed in the nucleus;EGFP and mCherry proteins could be expressed simultaneously in the cytoplasm;③The optimum voltages for Hela, 293T and 3T3 cells were 425 V, 400 V and 440 V respectively, and the optimal pulse time was 30 ms. Conclusion This experiment demonstrates:①The feasibility of transfection of modRNA into cytoplasm and nucleus by electroporation;②Simultaneous introduction of two modRNAs into the same cell and expression;③ Optimal voltage and optimal pulse time are based on different cell types.

关 键 词：合成修饰的信使核糖核酸 电转法 最适电压 最适脉冲时间 

分 类 号：Q813.11[生物学—生物工程]