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

作　　者：张爱燕 陈程 吴晓丹 任天丽[3] 顾兵 黄红宇[2] 韩志君[2] 高明珠[2] 

机构地区：[1]徐州医科大学医学技术学院,江苏徐州221004 [2]南京医科大学附属无锡第二人民医院检验科,江苏无锡214002 [3]南京医科大学附属无锡第二人民医院风湿科,江苏无锡214002

出　　处：《临床检验杂志》2017年第8期583-586,共4页Chinese Journal of Clinical Laboratory Science

基　　金：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)

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Exo QuickTM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(×10~3)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。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 Exo QuickTMkit. The morphological characteristics and particle size of exosomes were determined by transmission electron microscope（ TEM） and Nano Sight 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 55-70 k D in both PM/DM patients and healthy controls,and that there were different bands in 40-55 k D 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.

关 键 词：多发性肌炎/皮肌炎 外泌体 分离 鉴定 

分 类 号：R446.61[医药卫生—诊断学]