分泌包载ANXA2-MS2 RNA外泌体的稳转细胞株的构建

摘要 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。

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

ABSTRACT 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.