大鼠T细胞源性exosome提取方法比较

发布时间：2018-01-07 21:13

本文关键词：大鼠T细胞源性exosome提取方法比较　出处：《重庆医学》2013年29期 　论文类型：期刊论文

【摘要】：目的采用3种方法从大鼠T细胞培养上清中提取纯化exosome,以获取高质量的exosome。方法分别采用ExoQuick Precipitation提取法、超滤密度梯度离心法、差速离心法提取T细胞培养上清中的exosome。利用透射电镜进行形态学观察,2,2-联喹啉-4,4-二甲酸二钠(BCA)法进行蛋白定量,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析蛋白表达的差异,Western blotting检测白细胞介素2(IL-2)的表达。结果 3种方法均可提取出exosome;ExoQuick Precipitation提取法、超滤密度梯度离心法所得样本浓度显著高于差速离心法所得样本浓度(P0.05);SDS-PAGE结果显示3种方法所得样本具有蛋白表达强度的差异;Western blotting显示3种方法所得样本均表达IL-2。结论 ExoQuick Precipitation提取法、超滤密度梯度离心法可获得高纯度、无蛋白丢失的exosome样本。
[Abstract]:Objective to develop the extraction and purification of exosome in supernatant from rat T cells by 3 methods, in order to obtain high quality exosome. methods respectively using ExoQuick Precipitation extraction, ultrafiltration by density gradient centrifugation. The extraction of T in cell culture supernatant by exosome. transmission electron microscope was used to observe the morphology of differential centrifugation, 2,2- biquinolyl two formic acid and two -4,4- sodium (BCA) method for quantitative protein, twelve dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of differences in protein expression, Western blotting detection of interleukin 2 (IL-2) expression. Results of the 3 methods can extract exosome; ExoQuick Precipitation extraction method, the sample concentration was significantly higher than that by density gradient centrifugation and ultrafiltration differential centrifugation of the sample concentration (P0.05); SDS-PAGE results showed that the samples of the 3 kinds of methods with the difference of expression intensity of Western protein; blotting 3 Methods the samples were IL-2. expression conclusion ExoQuick Precipitation extraction, ultrafiltration and density gradient centrifugation with high purity can be obtained, without loss of protein exosome samples.

【作者单位】：第三军医大学新桥医院泌尿外科;
【基金】：国家自然科学基金资助项目(30872580)
【分类号】：R-332
【正文快照】： Exosome是一种起源于内吞体系统并被排出胞外的膜性囊泡,直径在30~100nm[1],含有大量与其来源相关的特异性蛋白,并由此发挥不同的功能[2]。T淋巴细胞是免疫系统的重要组成部分,并且不同的亚群具有各异的免疫学功能。例如CD4+CD25+Terg细胞具有诱导免疫耐受的作用[3];而接触抗

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