| 摘要: |
| 目的 通过对比不同方法抽提的不同组织中的细胞外囊泡的差异和特点，探寻组织中细胞外囊泡的分离纯化方法，为组织微环境中细胞外囊泡的功能和机制研究奠定基础。方法 采用胶原酶消化法对肾脏、前列腺、皮肤和胃4种组织进行脱细胞处理，制备细胞外基质悬液，分别采用Qiagen外泌体抽提试剂盒、SBI外泌体抽提试剂盒及超速离心法（UC）抽提细胞外囊泡，然后使用ATS粒度分析仪qNano技术检测细胞外囊泡的粒径和浓度等。结果 采用3种方法对4种组织的细胞外基质悬液进行抽提，均可获得纯化的细胞外囊泡，主要囊泡类型为外泌体。不同组织中细胞外囊泡粒径分布存在一定的差异，但粒径大小差异无统计学意义（P>0.05）。等质量组织中均以UC抽提得到的细胞外囊泡浓度最高，与另外2种方法相比差异有统计学意义（P<0.01）；不同组织间比较，以前列腺和肾脏获得的细胞外囊泡浓度较高，而胃和皮肤较低，差异有统计学意义（P<0.05）。结论 不同组织中均以UC抽提得到的细胞外囊泡浓度最高；不同类型组织微环境中存在的细胞外囊泡的浓度有差异。 |
| 关键词: 细胞外囊泡 外泌体 组织微环境 纯化 |
| DOI：10.16781/j.0258-879x.2018.07.0726 |
| 投稿时间：2017-12-23修订日期：2018-05-16 |
| 基金项目:上海市青年科技启明星计划（17QA1405400），上海市卫生计生系统优秀青年医学人才培养计划（2017YQ028）. |
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| Separation and purification of extracellular vesicles in tissue microenvironment and its physiochemical characteristics |
| LI Li1,ZENG Tao1,YE Yu-sheng2,WANG Yue1,3* |
(1. Department of Histology and Embryology, College of Basic Medical Sciences, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
2. The Second Student Team, College of Basic Medical Sciences, Navy Medical University(Second Military Medical University), Shanghai 200433, China;
3. Center of Translational Medicine, Institute of Translational Medicine, Navy Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding author) |
| Abstract: |
| Objective To compare the differences and characteristics of extracellular vesicles in different tissues extracted by different methods, and to explore the separation and purification methods of extracellular vesicles in tissues, so as to lay a foundation for studying the function and mechanism of extracellular vesicles in tissue microenvironment. Methods Collagenase digestion was performed on four tissues:kidney, prostate, skin and stomach, to obtain decellularized tissues and to prepare extracellular matrix suspensions. Qiagen exosome extraction kit and SBI exosome extraction kit and ultracentrifugation (UC) method were used to extract the extracellular vesicles. The ATS particle size analyzer qNano technique was used to measure the particle size and concentration of the extracellular vesicles. Results Purified extracellular vesicles were obtained by the three methods from the extracellular matrix suspensions of the four tissues, and the main vesicle type was exosomes. The distribution of particle size was different among the different types of tissues, but the difference of particle size was not significant (P>0.05). In homogeneous tissues, the extracellular vesicles obtained by UC method had the highest concentration, which were significantly different compared with the concentrations of the extracellular vesicles obtained by the other two methods (P<0.01). The concentrations of the extracellular vesicles from prostate and kidney were higher, and those from stomach and skin were lower, and the difference was statistically significant (P<0.05). Conclusion The concentration of extracellular vesicles extracted by UC method is the highest in the various types of tissue microenvironments. There is a significant difference in the concentrations of extracellular vesicles from different types of tissue microenvironments. |
| Key words: extracellular vesicles exosomes tissue microenvironment purification |
