分泌包载ANXA2-MS2 RNA外泌体的稳转细胞株的构建

Cell Line Construction of a Stable ANXA2-MS2 RNA Expression in Exosomes

目的:基于MCP-MS2示踪系统构建能稳定分泌含有标签的膜联蛋白A2(ANXA2)RNA的外泌体的供体细胞株。方法:利用慢病毒载体构建稳定表达MCP-EGFP的HEK293细胞株,采用流式细胞技术筛选具有较好荧光特性的单克隆细胞株。然后再次通过慢病毒稳定表达系统构建出能分泌结合有MCP-EGFP标记的ANXA2 RNA外泌体的供体细胞株。通过透射电镜、NTA、NanoFCM、共聚焦显微镜等仪器对供体细胞所产生的外泌体进行表征。结果:通过慢病毒载体成功构建了MCP-EGFP稳定表达细胞株,然后利用该细胞株通过慢病毒载体成功导入含有MS2标签的ANXA2基因。实验结果表明,构建的供体细胞与正常细胞所分泌的外泌体形态相似,实实施时荧光定量PCR的结果显示分泌的外泌体中含有MS2特殊序列,而同时Western blot的结果显示分泌的外泌体中含有EGFP蛋白。结论:通过慢病毒法成功建立了能分泌含有MS2标记的ANXA2 RNA的外泌体供体细胞株,为高效、稳定的装载特定RNA外泌体的供体细胞株的构建提供了新的方法和理论基础。

Objective:To construct a donor cell line which can stably produce exosomes containing MS2 labeled ANXA2 RNA based on the MCP-MS2 system.To explore an efficient construction method of a stable donor cell line containing specific RNA labeled exosomes.Methods:The HEK293 cell line stably expressing MCP-EGFP was constructed by lentivirus transfection system,and the monoclone cell line with better fluorescence characteristics was screened by flow cytometry.Then,a donor cell line,that secrete ANXA2 RNA exosomes labeled with MCP-EGFP,was constructed by lentivirus stable expression system.The exosomes production were characterized by transmission electron microscopy,NTA,NanoFCM,confocal microscopy and other instruments.Results:The stable expression cell line of MCP-EGFP was successfully constructed by lentivirus vector,and then the ANXA2-MS2 gene was successfully introduced into the cell line by the same method.The exosomes obtained from the constructed donor cells were similar to those secreted by normal cells.RT-PCR showed that the exosomes also contained the MS2 sequence,while Western blot showed that the exosomes contained the EGFP protein expression.Conclusion:A donor cell line,which can produce exosomes containing ANXA2 RNA with MS2 tag,was successfully established by lentivirus system.This research provides a new method and theoretical basis for the construction of a donor cell line that can efficiently and stably loading specific RNA in exosomes.