HK-2外泌体提取及体外细胞靶向性的研究  

Extraction of HK-2 exosomes and cell targeting in vitro

作　　者：谭小宁[1] 李勇敏[1] 马荣丽[2] 罗吉[1] 吕元[1] TAN Xiaoning;LI Yongmin;MA Rongli;LUO Ji;LV Yuan(Hunan Academy of Traditional Chinese Medicine Affiliated Hospital,Changsha,Hunan 410006,China;Hunan University of Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]湖南省中医药研究院附属医院,湖南长沙410006 [2]湖南中医药大学,湖南长沙410208

出　　处：《湖南中医药大学学报》2022年第6期923-929,共7页Journal of Hunan University of Chinese Medicine

基　　金：国家自然科学基金面上项目(81774163);湖南省自然科学基金项目(2018JJ6023,2020JJ5327);湖南省科技创新平台与人才计划重点实验室(2017TP1033);湖南省临床医疗技术创新引导项目(2017SK50404);长沙市科技计划项目(kq1901065)。

摘　　要：目的比较HK-2外泌体分离方法对粒径分布的影响,探索体外细胞摄取外泌体模式对外泌体细胞靶向性的参考价值。方法采用超速离心法和3种试剂盒分离HK-2外泌体,测定其粒径分布;用PKH67标记HK-2外泌体,体外观察HFL1、hFOB1.19、Kupffer、HCT1164种细胞吸收HK-2外泌体,以及HCT116吸收HCT116、HK-2、HFL1、Kupffer 4种细胞的外泌体情况。结果不同的分离方法得到外泌体的粒径分析结果显示,超速离心法得到外膜囊泡的粒径比较均一,集中在100 nm左右。体外细胞吸收外泌体试验结果显示:(1)在体外,HK-2外泌体均能被HFL1、hFOB1.19、Kupffer、HCT116细胞吸收;(2)HCT116能吸收HCT116、HK-2、HFL1、Kupffer细胞的外泌体。结论超速离心法和试剂盒都能分离得到HK-2外泌体,超速离心法得到外泌体粒径大小分布较均一;试剂盒操作比较便捷,但是大囊泡和杂蛋白居多。在体外,同种外泌体能被多种细胞摄取吸收,同种细胞能吸收不同种细胞外泌体,表明外泌体在体外没有特别好的靶向性。Objective To compare the effect of HK-2 exosomes separation method on particle size distribution,and to explore the reference value of model of exosome uptake by cells in vitro for exosome targeting.Methods HK-2 exosomes were separated by supercentrifugation and three kits,and its particle size distribution was determined.HK-2 exosomes were labeled with PKH67 to observe the absorption by HFL1,hFOB1.19,Kupffer and HCT116 cells,and exosomes of HCT116,HK-2,HFL1 and Kupffer cells were absorbed by HCT116.Results The particle size analysis of exosomes obtained by different separation methods showed that the particle size of outer membrane vesicles obtained by supercentrifugation was relatively uniform,concentrated at about 100 nm.The results of in vitro cell absorption of exosomes showed that:(1)HFL1,hFOB1.19,Kupffer and HCT116 cells could absorb HK-2 exosomes in vitro.(2)HCT116 can absorb exosomes of HCT116,HK-2,HFL1 and Kupffer cells.Conclusion HK-2exosomes can be isolated by supercentrifugation and kit.The particle size distribution of the exosomes obtained by supercentrifugation method is relatively uniform.The kit is relatively easy to operate,but large vesicles and miscellaneous proteins are in the majority.In vitro,the same exosomes can be absorbed by multiple cells,and the same cells can absorb different kinds of exosomes,indicating that there is no particularly good targeting.

关 键 词：外泌体 超速离心法 粒径分布 体外细胞 靶向吸收 

分 类 号：R2[医药卫生—中医学]