张爱燕,陈程,吴晓丹,任天丽,顾兵,黄红宇,韩志君,高明珠.多发性肌炎/皮肌炎患者血清中外泌体的分离鉴定[J].临床检验杂志,2017,(8):583-586
多发性肌炎/皮肌炎患者血清中外泌体的分离鉴定
Isolation and identification of serum exosomes in the patients with polymyositis/dermatomyositis
投稿时间:2017-03-15  
DOI:
中文关键词:  多发性肌炎/皮肌炎  外泌体  分离  鉴定
英文关键词:polymyositis/dermatomyositis  exosome  isolation  identification
基金项目:国家自然科学基金(81501503);无锡市卫计委医学科研项目(YGZXM1501);南京医科大学科技发展基金面上项目(2014NJMU062)。
作者单位
张爱燕 徐州医科大学医学技术学院江苏徐州 221004 
陈程 徐州医科大学医学技术学院江苏徐州 221004 
吴晓丹 南京医科大学附属无锡第二人民医院检验科江苏无锡 214002 
任天丽 南京医科大学附属无锡第二人民医院风湿科江苏无锡 214002 
顾兵 徐州医科大学医学技术学院江苏徐州 221004 
黄红宇 南京医科大学附属无锡第二人民医院检验科江苏无锡 214002 
韩志君 南京医科大学附属无锡第二人民医院检验科江苏无锡 214002 
高明珠 南京医科大学附属无锡第二人民医院检验科江苏无锡 214002 
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中文摘要:
      摘要:目的:从多发性肌炎/皮肌炎(polymyositis/dermatomyositis,PM/DM)患者外周血血清中分离鉴定外泌体(exosome),并进行初步的蛋白质研究。 方法:用ExoQuickTM试剂盒分离纯化PM/DM患者血清exosome;透射电镜观察其形态特征,Nanosight可视型纳米颗粒分析仪检测粒径大小分布情况;western blot鉴定exosome的表面标志物CD9,CD81,Flotillin-2;BCA法对其所携带的蛋白质进行定量分析;十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)初步分析其蛋白质组分。 结果:〓透射电镜下患者血清exosome呈圆形或椭圆形膜性小囊泡,直径分布范围92±67 nm;western blot结果表明,PM/DM血清来源的exosome存在表面标志物CD9、CD81、Flotillin-2;BCA法测定PM/DM患者exosome总蛋白浓度为14.68(6.00,32.55)μg/μL,健康组exosome总蛋白浓度为14.09(8.00,23.28)μg/μL,SDS PAGE显示两组血清exosome均在Mr(×103)为55~70处有高丰度蛋白质富集,在Mr(×103)为40~55处存在差异条带。 结论:成功从PM/DM患者的外周血中分离出血清exosome,并为寻找差异蛋白质提供实验依据。
英文摘要:
      Abstract: Objective:To isolate and identify exosomes from serum samples of the patients with polymyositis / dermatomyositis (PM/DM), and analyze their protein composition preliminarily. Methods:Exosomes from serum samples of the patients with PM/DM were isolated and purified by the ExoQuickTM kit. The morphological characteristics and particle size of exosomes were determined by transmission electron microscope (TEM) and NanoSight analyzer, respectively. The surface markers of exosomes such as CD9, CD81 and Flotillin 2 were identified by western blot. The concentration and composition of exosome protein were determined by the BCA method and SDS PAGE, respectively. Results:The exosomes from serum samples of PM/DM patients displayed round or oval vesicles with membrane structure under TEM, and their diameter range was about (92±67) nm. western blot showed that these exosomes expressed CD9, CD81 and Flotillin-2. The total protein concentrations of exosomes in the patients with PM/DM and healthy controls were 14.68 (6.00,32.55) μg/μL and 14.09 (8.00,23.28) μg/μL, respectively. SDS-PAGE showed that high abundance proteins enriched in 5570 kD in both PM/DM patients and healthy controls, and that there were different bands in 4055 kD between them. Conclusion:Exosomes are isolated from serum samples of the patients with PM/DM successfully, and their protein concentration and composition are analyzed preliminarily, which provides the experimental evidences for further finding differential proteins.
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