GW4869抑制骨髓间充质干细胞外泌体的释放  

GW4869 inhibits the release of exosomes from bone marrow mesenchymal stem cells

作　　者：孙淑丽[1] 肖培欣 丁辉[2] 王敬[1] 刘子泉 刘晋阳[1] 王雪[1] 石沙[3] 吕琪 樊毫军[4] Sun Shu-li;Xiao Pei-xin;Ding Hui;Wang Jing;Liu Zi-quan;Liu Jin-yang;Wang Xue;Shi Sha;Lv Qi;Fan Hao-jun(institute of Disaster Medicine and Public Health,Tianjin 300162,China;lnstitute of Disaster Medicine,Tianjin University,Tianjin 300072,China;Student Brigade 2,Logistic University of Chinese People's Armed Police Force,Tianjin 300309,China;Division of Medical Teaching,Affiliated Hospital of Logistic University of Chinese People's Armed Police Force,Tianjin 300162,China)

机构地区：[1]武警后勤学院附属医院救援医学研究所,天津市300162 [2]天津大学突难医学研究院,天津市300072 [3]武警后勤学院学员2旅,天津市300309 [4]武警后勤学院附属医院医教部,天津市300162

出　　处：《中国组织工程研究》2018年第1期26-31,共6页Chinese Journal of Tissue Engineering Research

基　　金：天津市科技计划项目(15ZXLCSY00040)；天津市应用基础与前沿技术研究计划(15JCYBJC28500)；天津市卫生计生委科技基金项目(2015KZ125)

摘　　要：背景:间充质干细胞来源外泌体与间充质干细胞本身具有相似的功能,但外泌体释放的调控机制依然不是很清楚。目的:探讨中性鞘磷脂酶抑制剂GW4869对骨髓间充质干细胞来源外泌体释放的抑制作用及其对细胞增殖的影响。方法:将骨髓间充质干细胞分为正常对照组,GW4869处理24 h组和GW4869撤药24 h组(GW4869预处理24 h,撤药后继续培养24 h),分别收集各组条件培养基,采用超速离心法提取外泌体,Western blot法鉴定外泌体表面标志蛋白CD63、TSG101的表达,Nano-sight检测外泌体粒径分布及浓度,BCA法测定外泌体总蛋白水平,活细胞成像系统观察GW4869对骨髓间充质干细胞增殖的影响。结果与结论:(1)Western blot显示外泌体阳性表达标志蛋白CD63、TSG101;(2)纳米示综分析证实释放的外泌体大小约114 nm;(3)与正常对照组相比,GW4869处理24 h组和GW4869撤药24 h组外泌体释放减少差异有显著性意义(P<0.01),而GW4869预处理24 h组和GW4869撤药24 h组差异无显著性意义(P>0.05);(4)与正常对照组相比,GW4869及其溶剂二甲基亚砜对骨髓间充质干细胞增殖影响差异无显著性意义(P>0.05);(5)结果表明,GW4869可以显著抑制骨髓间充质干细胞外泌体的释放,并且在撤药24 h内依然保持抑制作用,GW4869对骨髓间充质干细胞增殖无影响。BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)and BMSCs-derived exosomes have similar functions,but the regulatory mechanism underlying the release of exosomes is still unclear.OBJECTIVE:To investigate the role of GW4869,an inhibition of neutral sphingomyelinase 2,in the release of exosomes in BMSCs and the influence of GW4869 on BMSCs proliferation.METHODS:Rat BMSCs were divided into three groups:normal control group,24-hour GW4869 treatment group and withdrawal of GW4869 for 24 hours group(24-hour GW4869 treatment followed by 24-hour successive culture with drug withdrawal).Cultured cells were collected to extract exosomes by ultracentrifugation.Western blot was used to detect exosome-associated proteins CD63 and tumor susceptibility gene 101(TSG101).The concentration and size distribution of exosomes were measured using nanoparticle tracking analysis.BCA was used to test the level of total proteins in exosomes.Live cell imaging system was used to observe the influence of GW4869 on BMSCs proliferation.RESULTS AND CONCLUSION:(1)Western blot results showed that exosomes expressed marker proteins such as CD63,TSG101.(2)Findings from the nanoparticle tracking analysis confirmed that the size of released exosomes was about 114 nm.(3)Significantly reduced release of exosomes was found in the two treatment groups compared with the normal control group(P<0.01),but there was no significant difference between 24-hour GW4869 treatment group and withdrawal of GW4869 for 24 hours group(P>0.05).(4)No significant difference in the proliferation of BMSCs was found among the three groups(P>0.05).To conclude,24-hour W4869 can inhibit the release of exosomes by BMSCs and this inhibitory effect is still sustained within 24 hours after drug withdrawal.However,GW4869 has no influence on the proliferation of BMSCs.

关 键 词：骨髓 间质干细胞 外泌体 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]