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

作　　者：李凯祥 罗润娇 于新迪[1] 刘明璐 卢婷婷 王湘莹 王会景 付炜[2,3] 王伟 LI Kaixiang;LUO Runjiao;YU Xindi;LIU Minglu;LU Tingting;WANG Xiangying;WANG Huijing;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胸心外科,上海市200127 [2]上海交通大学医学院附属上海儿童医学中心儿科转化医学研究所,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》2024年第3期275-284,共10页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家自然科学基金(82070430,82272178);上海交通大学“交大之星”计划医工交叉研究基金(YG2021GD04);上海市浦东新区科技发展基金(PKJ2020-Y06)。

摘　　要：目的筛选更优的转染方式,探讨其用于modRNA转染人脂肪干细胞(Human adipose-derived stem cell,hADSC)并表达特定蛋白的可行性。方法分别采用2种电转染方式Optimization4及Optimization6和脂质体转染试剂RNAiMAX,将modGFP转染到hADSC中去。应用流式细胞分析仪和荧光显微镜分析,比较不同转染方式的转染效率和蛋白表达情况;通过细胞死活染色,评估转染后24 h细胞的存活情况;将更优的电转方式与RNAiMAX用于modVEGFA转染hADSC,收集其培养24 h的上清用于人脐静脉内皮细胞(Human umbilical vein endothelial cells,hUVECs)成管和transwell迁移实验,并与基质胶混合后注射于裸鼠皮下,通过大体观、HE染色及免疫荧光染色,分析其在体内促进血管再生的情况。结果Optimization6电转转染hADSC的效率为87.75%±1.52%,高于Optimization4转染组(80.85%±1.48%)和RNAiMAX转染组(81.80%±1.08%),且荧光显微镜观察及流式分析均表明电转染方式的蛋白表达优于RNAiMAX脂质体转染。3种转染方式对细胞的毒性均较小,无统计学差异。与RNAiMAX相比,Optimization6电转能更高效地介导modVEGFA转染hADSC,能更好地促进体外hUVECs成管和迁移,皮下血管再生的效果也更佳。结论相比于RNAiMAX和Optimization4电转染,Optimization6电转方式转染hADSC的效率更高,蛋白表达高效,且能在体内外快速高效促进血管再生。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 

分 类 号：R318.02[医药卫生—生物医学工程]