摘要
目的探讨i PSC-MSC来源的外泌体（exosome,Exo）对LPS刺激的肺泡巨噬细胞释放炎性因子的作用。方法采用旋转超滤法提纯i PSC-MSC外泌体,以无外泌体培养基培养肺泡巨噬细胞NR8383,分别给予Exo（50μg/ml）、LPS（50ng/ml）、LPS（50ng/ml）＋Exo（50μg/ml）培养24h,以不含Exo和LPS培养为对照组,利用酶联免疫标记法（ELISA）检测各组上清液中TNF-α、IL-1β、IL-6蛋白浓度。结果提取物经透射电镜观察为圆形或半圆形囊泡,直径40-100nm,表达Exo标志物CD9和CD63;LPS组上清液中TNF-α、IL-1β和IL-6的浓度（435.38±36.31pg/ml、319.76±39.14pg/ml和408.33±43.44pg/ml）与空白对照组（37.48±8.75pg/ml、33.51±7.88pg/ml和37.73±8.46pg/ml）和Exo组（38.71±9.14pg/ml、32.05±6.81pg/ml和42.84±6.54pg/ml）比较显著上调（P〈0.01）;LPS＋Exo组TNF-α、IL-1β和IL-6的浓度（369.30±32.74pg/ml、249.23±36.77pg/ml和328.91±46.45pg/ml）与LPS组相比,明显减少（P〈0.05）;空白对照组与Exo组的TNF-α、IL-1β和IL-6的浓度无显著性差异。结论成功富集Exo;i PSC-MSC来源的Exo可抑制LPS诱导的肺泡巨噬细胞表达炎性因子。
Objective To investigate the effect of exosomes secreted by i PSC-MSC on the inflammatory factors release of alveolar macrophage induced by LPS. Methods Exosome（Exo） was isolated from supernatant culture medium of i PSCs-MSCsby ultrafiltration method. Alveolar macrophage NR8383 was cultured with exosome-free fetal bovine serum,Exo（50μg/ml）,LPS（50ng/ml）,LPS（50ng/ml）＋Exo（50μg/ml） were added into the culture medium, and these three culture manners were defined as Exo group,LPS group and LPS＋Exo group. After 24 h,the TNF-α,IL-1βand IL-6 of the culture supernatant were detected by ELISA. Result Rounded and oval vesicles were observed by transmission electron microscope,and the diameter was about 40-100 nm. CD9 and CD63 proteins were detected on the vesicles＇ membrane. In LPS group,the concentrations of TNF-α,IL-1β and IL-6（435.38±36.31,319.76±39.14 and 408.33±43.44pg/ml） were higher than those in control group（37.48±8.75,33.51±7.88 and 37.73±8.46pg/ml）and Exo group（38.71±9.14,32.05±6.81 and 42.84±6.54pg/ml）（P0.01）. Compared with LPS group,the concentrations of TNF-α,IL-1β and IL-6（369.30±32.74,249.23±36.77 and 328.91±46.45 pg/ml） were significantly reduced in LPS＋Exo group（P0.05）;there was no difference between them in control group and Exo group. Conclusion Exosomes were successfully enriched,and exosomes derived from i PSC-MSC can alleviate the inflammation effect of alveolar macrophages induced by LPS.
出处
《实验与检验医学》
CAS
2016年第1期4-7,共4页
Experimental and Laboratory Medicine
基金
国家自然科学基金(81460292)、国家自然科学基金(81560306)
关键词
外泌体
肺泡巨噬细胞
脓毒症肺损伤
炎症因子
旋转超滤法
Exosomes
Alveolar macrophage
Sepsis-associated lung injury
Inflammatory factor
Stirring ultrafiltration
