组织工程与重建外科杂志2024，Vol.20Issue(3)：275-284,10.DOI:10.3969/j.issn.1673-0364.2024.03.001
不同转染方式用于modRNA转染人脂肪干细胞的初步研究
Preliminary study on modRNA transfection of human adipose-derived stem cells with different transfection methods
摘要
Abstract
Objective To select a transfection method with better efficiency and to determine its feasibility in modRNA transfection of human adipose-derived stem cell(hADSC)as well as specific protein expression pattern.Methods ModGFP was transfected into hADSC using two kinds of electrotransfection methods(Optimization4 and Optimization6)and liposome transfection reagent(RNAiMAX).The transfection efficiency and protein expression of different transfection methods were analyzed by flow cytometry and fluorescence microscope.Cell survival rate at 24 h after transfection was evaluated by AM/PI staining.Electrotransformation mode with better effect and RNAiMAX were used to transfect modVEGFA into hADSC.Culture supernatant of hADSC after being transfected for 24 h was collected to incubate human umbilical vein endothelial cells(hUVECs)followed by tube formation and transwell migration experiments.hADSCmodVEGFA mixed with matrigel was injected subcutaneously in nude mice.The ability to stimulate angiogenesis in vivo was analyzed by gross observation,HE staining,and immunofluorescence staining.Results The transfection efficiency was higher in Optimization6 group(87.75%±1.52%),compared with Optimization4 group(80.85%±1.48%)and RNAiMAX group(81.8%±1.08%).Fluorescence microscopy and flow analysis showed that the protein expression pattern was better in electrotransfection groups than that of RNAiMAX group.The toxicity of these three transfection methods in cells was minimal,no statistical difference was found.Compared with RNAiMAX,Optimization6 can transfect modVEGFA more efficiently into hADSC and possess better ability of promoting angiogenesis both in vitro and in vivo.Conclusion Compared with RNAiMAX and Optimization4,Optimization6 has higher transfection efficiency,better protein expression pattern,and stronger angiogenesis promoting effect both in vitro and in vivo.关键词
化学合成修饰信使核糖核酸/基因表达/电转染/脂质体/人脂肪干细胞Key words
Chemically synthetic modified message RNA/Gene expression/Electroporation/Liposome/Human adipose-derived stem cell分类
医药卫生引用本文复制引用
李凯祥,罗润娇,于新迪,刘明璐,卢婷婷,王湘莹,王会景,付炜,王伟..不同转染方式用于modRNA转染人脂肪干细胞的初步研究[J].组织工程与重建外科杂志,2024,20(3):275-284,10.基金项目
国家自然科学基金(82070430,82272178) （82070430,82272178）
上海交通大学"交大之星"计划医工交叉研究基金(YG2021GD04) （YG2021GD04）
上海市浦东新区科技发展基金(PKJ2020-Y06). （PKJ2020-Y06）
