摘要
外泌体是一种细胞分泌的囊泡,里面含有丰富的核酸和蛋白质等物质,对于信息的传递、免疫功能的调节具有重要作用.本研究通过培养巨噬细胞、树突状细胞,收集上清液,分别采用外泌体提取试剂盒法和超速离心法提取外泌体,使用透射电镜法和Western blot法进行外泌体鉴定.将提取的外泌体转染荧光标记,与淋巴细胞共同孵育24 h.采用含荧光标记的miR-155,分别转染到巨噬细胞和树突细胞中,提取细胞上清的外泌体,与淋巴细胞共孵育24 h.结果显示:超速离心法提取的外泌体更纯,但是SBI试剂盒提取的外泌体更多,适用于下一步试验;巨噬细胞及树突状细胞分泌的外泌体加荧光标记与淋巴细胞共培养后,可使淋巴细胞荧光增加;荧光标记miR-155的外泌体与淋巴细胞共培养,可使淋巴细胞荧光增加.本研究证实了树突细胞、巨噬细胞分泌的外泌体及其包含的miR-155可作用于淋巴细胞,参与免疫细胞间的通讯,为进一步阐明外泌体的作用机制奠定了理论基础.
Exosomes are vesicles secreted by cells,rich in nucleic acids and proteins,which play an important role in the transmission of information and the regulation of immune function.In this study,macrophages and dendritic cells were cultured,the supernatant was collected,and the exosomes were extracted by the kit method and ultracentrifugation method,and the exosomes were identified by the TEM electron microscope method and the Western blot method.The extracted exosomes were transfected with fluorescent markers and incubated with lymphocytes for 24 h;miR-155 containing fluorescent markers was used to transfect macrophages and dendritic cells respectively,and the exosomes from the cell supernatant were extracted and combined with the lymphocytes and were incubated together for 24 h.The results showed that the exosomes extracted by ultracentrifugation were more pure,but the exosomes extracted by the SBI kit were more suitable for the next experiment.After the exosomes secreted by macrophages and dendritic cells were co-cultured with lymphocytes with fluorescent labels,the fluorescence of lymphocytes increased.Co-culture of fluorescently labeled miR-155 exosomes and lymphocytes can increase the fluorescence of lymphocytes.This study confirmed that the exosomes secreted by dendritic cells and macrophages and the miR-155 contained therein can act on lymphocytes and participate in the communication between immune cells,laying a theoretical foundation for further elucidating the mechanism of exosomes.
作者
李婉雁
相雪莲
曹楠
许丹宁
陈文斌
张续勐
沈栩
黄运茂
田允波
LI Wanyan;XIANG Xuelian;CAO Nan;XU Danning;CHEN Wenbin;ZHANG Xumeng;SHEN Xu;HUANG Yunmao;TIAN Yunbo(College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Guangdong Province Key Laboratory of Waterfowl Healthy Breeding,Guangzhou 510225,China)
出处
《仲恺农业工程学院学报》
CAS
2021年第3期17-22,共6页
Journal of Zhongkai University of Agriculture and Engineering
基金
广东省基础与应用基础研究基金项目区域联合基金-青年基金项目(2019A1515110106)资助.
