药学学报, 2019, 54(9): 1688-1694
引用本文:
刘玉萍, 傅荣萍, 陈彦, 屠书梅. 大蒜素二烯丙基三硫醚-外泌体抗小鼠黑色素瘤肺转移的作用[J]. 药学学报, 2019, 54(9): 1688-1694.
LIU Yu-ping, FU Rong-ping, CHEN Yan, TU Shu-mei. Anti-metastatic effect of diallyl trisulfide-exosome in mouse melanoma[J]. Acta Pharmaceutica Sinica, 2019, 54(9): 1688-1694.

大蒜素二烯丙基三硫醚-外泌体抗小鼠黑色素瘤肺转移的作用
刘玉萍1,2, 傅荣萍1,2, 陈彦1,2, 屠书梅1,2
1. 南京中医药大学附属中西医结合医院, 江苏 南京 210028;
2. 江苏省中医药研究院, 中药组分与微生态研究中心, 江苏 南京 210028
摘要:
本研究从肺转移小鼠黑色素瘤细胞B16BL6中提取外泌体(exosome,Exo),制备了大蒜素二烯丙基三硫醚(diallyl trisulfide,DATS)含药外泌体(DATS-Exo)载药系统,探索该载药系统的抗肿瘤转移作用。采用差速超速离心法提取B16BL6细胞上清液中外泌体,并对外泌体的形态、粒径及电位进行表征,采用Western blot法检测外泌体特异分子CD9、TSG101、Flotillin 1及肺亲和蛋白α6的表达水平。应用超声方法制备DATS-Exo载药系统并进行表征,计算载药量。采用荧光显微镜及流式细胞术考察B16BL6对外泌体的摄取情况。采用经典的划痕实验法,评价DATS-Exo的体外抗迁移能力。建立小鼠黑色素瘤肺转移模型,动物实验获得南京中医药大学伦理委员会的同意,考察DATS-Exo的体内组织分布情况及抗肿瘤转移作用。结果表明,制备的DATS-Exo粒径为112.3 ±1.98 nm、多分散系数为0.24 ±0.08、zeta电位为-24.33 ±4.11 mV,呈茶托样结构。测得其蛋白浓度为1 312.33 ±6.27 μg·mL-1。DATS-Exo的载药量为0.33%±0.02%。B16BL6细胞能够摄取DATS-Exo,有效抑制B16BL6细胞的迁移,效果均强于DATS。DATS-Exo能够在肺组织聚集,并具有显著抑制肺转移作用。本研究结果表明,利用肺转移肿瘤细胞衍生的外泌体具有肺亲和性,并且具有载药性能,所制备的含药外泌体能够实现抑制肿瘤转移。
关键词:    外泌体      二烯丙基三硫醚      黑色素瘤      肿瘤转移      靶向     
Anti-metastatic effect of diallyl trisulfide-exosome in mouse melanoma
LIU Yu-ping1,2, FU Rong-ping1,2, CHEN Yan1,2, TU Shu-mei1,2
1. Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, China;
2. Multi-component of Traditional Chinese Medicine and Microecology Research Center, Jiangsu Provincial Academy of Chinese Medicine, Nanjing 210028, China
Abstract:
This study aimed to prepare an anti-metastatic diallyl trisulfide-exosome (DATS-Exo) drug delivery system. Exosomes in the supernatants of lung metastasis mouse melanoma B16BL6 cell line culture were extracted by ultracentrifugation. The quantity of exosomes was determined by transmission electron microscopy (TEM), Malvern particle size meter, and BCA assay. Expression levels of exosome-specific biomarkers CD9, TSG101, Flotillin 1 and lung organotropic biomarker α6 were detected by Western blot. The sonication method was used to load DATS into exosomes. The uptake of exosomes by B16BL6 cells and lung tissue was observed by laser confocal microscopy. Wound healing assay was used to evaluate the anti-migration effect of DATS-Exo in vitro. Experimental lung metastasis model was established to evaluate the anti-metastasis effect of DATS-Exo in vivo. Animal experiments have been approved by the Ethics Committee of Nanjing University of Chinese Medicine. The results showed that the particle size of DATS-Exo was 112.3 ±1.98 nm, polydispersity index (PDI) was 0.24 ±0.08, zeta potential was -24.33 ±4.11 mV, and the particles have classic tea tray-like membrane structure under TEM. The protein concentration of DATS-Exo was measured to be 1 312.33 ±6.27 μg·mL-1. The drug loading rate was about 0.33% ±0.02%. The exosomes could be taken up by B16BL6 cells and the lung tissue. Compared with the free DATS group, DATS-Exo had a better inhibitory effect on tumor metastasis in vitro and in vivo. Taken together, these results indicate that exosomes derived from the lung metastasis tumor cells have lung organotropic characteristic and drug-loading properties. Using these kind of exosomes as carrier for anti-tumor drug delivery can be a novel and effective strategy of anti-metastatic therapy.
Key words:    exosome    diallyl trisulfide    melanoma    metastasis    targeting   
收稿日期: 2019-06-25
DOI: 10.16438/j.0513-4870.2019-0511
基金项目: 江苏省自然科学基金青年基金(BK20171097);江苏省中医药局科技项目(YB2017033);江苏省科教强卫医学重点人才项目(ZDRCA2016036).
通讯作者: 陈彦,Tel:86-25-85608672,E-mail:ychen202@hotmail.com
Email: ychen202@hotmail.com
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