摘要
目的:探究多聚甲醛固定外泌体对外泌体本身、细胞活性和功能的影响,为研究外泌体相关策略选择提供基础。方法:采用透射电镜和Western blot鉴定外泌体,通过外泌体转染HepG2、Panc-1、Hela细胞后的形态和功能变化,鉴定多聚甲醛固定和未固定DIOC18(3)(3,3'-二十八烷氧碳菁高氯酸盐)染色对外泌体生物学特性的影响。采用CCK-8、MTT试验、细胞凋亡试验、细胞分泌外泌体的功能和细胞本身的功能来确定多聚甲醛固定和未固定DIO染色外泌体对细胞活力和功能的影响。结果:多聚甲醛固定DIO染色外泌体转染细胞阳性率比多聚甲醛未固定组高(20.21±0.05)%(P<0.05),前者的凋亡率比后者高(12.65±0.01)%(P<0.05)。多聚甲醛未固定DIO染色外泌体转染的细胞存活率比多聚甲醛固定组高(22.84±0.16)%(P<0.05),前者外泌体分泌率比后者高(28.10±5.65)%(P<0.05)。多聚甲醛未固定DIO染色HepG2.2.15细胞上清来源的外泌体转染HepG2细胞后,HBV DNA拷贝数比多聚甲醛固定DIO染色外泌体组高3.33倍(P<0.05)。结论:多聚甲醛固定DIO染色外泌体对外泌体本身特性及示踪定位研究具有良好效果;多聚甲醛未固定DIO染色外泌体亦能达到一定的示踪目的,但更能准确评价外泌体对细胞功能调控的作用。
Objective:To investigate the effects of paraformaldehyde fixation of exosomes on exosomes themselves,cell viability and functions,to provide the foundation for the selection of exosome-related research strategies.Methods:After the exosomes were identified by transmission electron microscopy and Western blot,the effects of paraformaldehyde fixed and non-fixed DIOC18(3)(3,3'-DIOctadecyloxacarbocyanine perchlorate)staining on the biological properties of exosomes were identified by morphological and func-tional HepG2,Panc-1,Hela cell changes after exosome transfection.CCK-8,MTT assay,apoptosis assay,cell function in secreting exosomes and the function of cells themselves were used to determine the effect of paraformaldehyde fixed and non-fixed DIO-stained exosomes on cell viability and function.Results:The positive rate of transfected cells which were transfected with paraformaldehyde fixed DIO stained exosomes was(20.21±0.05)%higher than paraformaldehyde non-fixed group(P<0.05),and the apoptosis rate of the former was(12.65±0.01)%higher than the latter(P<0.05).The survival rate of transfected cells which were transfected with para-formaldehyde non-fixed exosomes was(22.84±0.16)%higher than paraformaldehyde fixed group(P<0.05),and the exosome secretion rate of the former was(28.10±5.65)%higher than that the latter(P<0.05).The copy number of HBV DNA in HepG2 cells which were transfected with paraformaldehyde non-fixed,DIO stained,and the exosomes derived from HepG2.2.15 culture medium was 3.33 times higher than paraformaldehyde fixed DIO stained exosomes(P<0.05).Conclusion:The paraformaldehyde fixation and DIO staining of exosomes demonstrated good results for the investigation of exosomes'own characteristics,tracing and localization of exosomes.DIO staining of exosomes without paraformaldehyde fixation can also achieve the goal of tracing to a certain degree,but it can evaluate more accurately the role of exosomes in regulating cell function.
作者
刘佳敏
杨艳红
彭彬
戢敏
徐旭
张自力
曾蓉
潘万龙
LIU Jiamin;YANG Yanhong;PENG Bin;JI Min;XU Xu;ZHANG Zili;ZENG Rong;PAN Wanlong(Institute of Basic Medicine and Forensic Medicine,North Sichuan Medical College,Nanchong 637000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2023年第7期1351-1357,共7页
Chinese Journal of Immunology
基金
2019年川北医学院国家自然科学基金预研项目(CBY19-YZ07)。
作者简介
刘佳敏,女,在读硕士,主要从事外泌体在乙型肝炎病毒中的研究,E-mail:2239318480@qq.com;通信作者及指导教师:潘万龙,男,博士,教授,主要从事HBV感染分子免疫机制及外泌体研究,E-mail:panwl@aliyun.com。
