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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

28卷

期数:

2022年04期

页码:

353-359

栏目:

外泌体专题

出版日期:

2022-07-20

文章信息/Info

Title:

Study on the effect of different lyoprotectants on exosomes

文章编号:

1006-8147（2022）04-0353-07

作者:

吴颖杰; 耿梦缘; 汪晶; 尹海芳

（天津医科大学基础医学院&医学技术学院，天津 300070）

Author(s):

WU Ying-jie;  GENG Meng-yuan; WANG Jing; YIN Hai-fang

（School of Medical Technology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）

关键词:

外泌体; 冻干; 海藻糖; 运输; 储存

Keywords:

exosome; lyophilization：trehalose; transportation; preservation

分类号:

R781.4

DOI:

-

文献标志码:

A

摘要:

目的：通过测试海藻糖等不同冻干保护剂，分析其对外泌体的完整性、分散度以及生物学功能的保护作用。方法：超速离心法收集293T细胞来源的外泌体，在冻干过程中加入50～200 mmol/L赖氨酸、2%和4%乳糖、2%海藻糖和5%菊粉等不同的冻干保护剂；通过透射电镜分析外泌体的颗粒形貌、分散度；利用纳米粒度分析仪分析外泌体粒径分布和数量；利用血浆外泌体诱导血管成管实验，检测冻干后血浆外泌体诱导血管生成的能力；免疫印迹分析储存不同时间的外泌体上的标志蛋白CD63和CD81的表达情况；利用激光扫描共聚焦分析不同储存时间的外泌体被C2C12细胞摄取的能力。结果：50、100、200 mmol/L赖氨酸可以维持冻干293细胞外泌体完整性，但是不同浓度赖氨酸都无法解决外泌体冻干时的聚集问题。5%菊糖在冻干外泌体的分散度上表现良好，但是造成外泌体膜破损。4%乳糖和2%海藻糖可以维持外泌体形貌完整并保持外泌体分散度，并且保护效果相当。2%海藻糖冻干后血浆来源外泌体能诱导血管内皮细胞成管。将外泌体用2%海藻糖冻干后室温储存1 d、1周和1个月，和-80 ℃组外泌体相比，冻干外泌体仍然保持原有的形貌和分散度，不会改变外泌体的粒径分布，外泌体总颗粒数也没有差异（P＞0.05），并且总颗粒数显著高于不加冻干保护剂组（F＝28.8，P＜0.001）。常温条件下储存1个月，和-80°C组外泌体相比，冻干外泌体标志蛋白CD63和CD81表达水平没有差异，而不加海藻糖的外泌体CD81和CD63蛋白表达水平明显下降。添加2%海藻糖冻干后的外泌体常温储存不同时间后依旧能被细胞摄取，不会影响外泌体作为纳米载体的进入细胞的固有属性。结论：2%海藻糖可作为外泌体的冻干保护剂，为外泌体的运输和长期储存提供可行的方案。

Abstract:

Objective： To investigate the effects of different lyophilization protectants，such as trehalose，on the integrity，dispersity，and biological functions of exosomes. Methods: 293T cell-derived exosomes were collected by ultracentrifugation，and different lyoprotectants such as 50-200 mmol/L lysine，2% and 4% lactose，2% trehalose，and 5% inulin were added during the lyophilization. The particle morphology and dispersion of exosomes were analyzed by transmission electron microscopy; the particle size distribution and quantity of exosomes were analyzed by nanoparticle size analyzer; Plasma exosome induced angiogenesis assay was used to detect the ability of plasma exosomes to induce angiogenesis after lyophilization. The expression of marker proteins CD63 and CD81 on exosomes stored at different times were analyzed by Western blotting. The ability of exosomes stored at different times taken up by C2C12 cells was analyzed by Laser scanning confocal. Results: 50，100 and 200 mmol/L lysine maintained the integrity of lyophilized 293 cell exosomes，but different concentrations of lysine could not solve the aggregation problem of exosomes during lyophilization. 5% inulin performed well in the dispersion of lyophilized exosomes，but caused damage to the exosome membrane. 4% lactose and 2% trehalose could maintain the integrity of exosome morphology and maintain exosome dispersion，and the protective effect was equivalent. Plasma-derived exosomes could induce vascular endothelial cells to form tubes after lyophilization with 2% trehalose. The exosomes were lyophilized with 2% trehalose and stored at room temperature for 1 day，1 week and 1 month. Compared with the exosomes in the -80℃ group，the lyophilized exosomes still maintained the original morphology and dispersion. There was no change in the particle size distribution of exosomes，and there was no difference in the total number of exosome particles（P>0.05），and the total number of particles was significantly higher than that in the group without lyoprotectant（F＝28.8，P<0.001）. After 1 month of storage at room temperature，the expression levels of lyophilized exosome marker proteins CD63 and CD81 were not different compared with the exosomes in the -80℃ group，while the exosomes without trehalose expressed CD81 and CD63 proteins level dropped significantly. The lyophilized exosomes added with 2% trehalose could still be taken up by cells after being stored at room temperature for different times，and did not affect the inherent properties of exosomes as nanocarriers entering cells. Conclusion: 2% trehalose can be used as an effective lyoprotectant for exosomes，providing a feasible solution for transport and long-term storage of exosomes.

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备注/Memo

备注/Memo:

作者简介:吴颖杰(1995-)，男，硕士在读，研究方向：外泌体储存；通信作者:尹海芳, E-mail: haifangyin@tmu.edu.cn。

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更新日期/Last Update: 2022-07-20