心脏干细胞分泌的exosome对氧化应激下心肌细胞的保护作用研究  

CPC derivedexosome protects cardiomyocytes from oxidative stress

作　　者：肖静[1,2] 潘宇[2] 李晓红[2] 姜霖[1,2] 杨翔宇[1,2] 吴岳恒[2] 雷和平[2] 余细勇[2] 

机构地区：[1]南方医科大学药学院,广东广州510515 [2]广东省人民医院广东省医学科学院,广东广州510080

出　　处：《中国药理学通报》2015年第12期1656-1660,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81330007;81120108003);广东省科技计划国际合作项目(No2014A050503047)

摘　　要：目的探索心脏干细胞分泌的外泌体(exosome)对体外氧化应激所致心肌细胞凋亡的保护作用。方法磁珠分选出Sca-1^+CPCs进行培养,流式细胞术鉴定;分离CPCs分泌的exosome,并用Western blot进行表型鉴定和用Nanosight进行粒径分析;免疫荧光法及PKH26标记技术观察心肌细胞H9c2对exosome的摄取作用;通过H_2O_2处理诱导H9c2细胞凋亡,用Western blot检测对照组与CPC-exo处理组凋亡相关蛋白水平,观察exosome对心肌细胞凋亡的保护作用。结果 1磁珠分选出的细胞用免疫荧光检测发现Sca-1呈阳性表达,且纯度可达95%以上。2从Sca-1^+CPCs细胞培养上清中提取出的exosome其表面标记蛋白CD63呈阳性表达,Nanosight结果显示大部分粒子都处于(82.33±3.06)nm(n=3)。免疫荧光显示H9c2细胞中可见大量PKH26标记的exosome在胞质聚集。3Western blot结果显示,与对照组相比,CPC-exo处理组总的caspase-3表达不变,而激活型caspase-3表达水平明显降低(P<0.05)。结论 CPCs分泌的exosome可被H9C2细胞所摄取,可以保护心肌细胞由于氧化应激引起的凋亡。Aim To explore the anti-apoptotic function of cardiac progenitor cells( CPCs)-derived exosome in vitro. Method CPCs were isolated from mouse heart using Magnetic Cell Sorting( MACS) system. Flow Cytometry( FC) determine the purity of stem cell surface antigen-1 positive( Sca-1+) CPCs. Exosome was purified from conditional medium,and confirmed by Western blot using CD63 as a marker,Nanoparticle Trafficking Analysis( NTA) was used to detect the diameters and concentration of exosome. Then the cells were divided into control groups and CPC-exosome pre-protection groups. H_2O_2 was added into H9c2 cells to induce oxidative stress. Western blot was adopted to determine the expression of cleaved caspase-3. Results 1 Immunofluorescence showed that CPCs isolated by MACS were positively expressing Sca-1 protein; FC analysis showed that typical purity of Sca-1+CPCs from the first preparations was more than 95%. 2 WB demonstrated that CD63 of exosome isolated from CCM was positively expressed,and NTA results showed that the diameters of exosome were( 82. 33 ± 3. 06) nm( n = 3). Microscope detected PKH-26 labeled exosome appeared in the cytoplasma of H9c2 cells. 3 Western blot showed the CPC-exosome pre-protection groups significantly down-regulated the levels of cleaved caspase-3 compared to the control groups( P < 0. 05). Conclusion CPC can secrete exosome which carries many important cargos,which can effectively gather in H9c2 cells.CPC-exosome can protect H9c2 cells from the oxidative stress induced by H2O2. Our results highlight a new perspective strategy for cardiac disease.

关 键 词：心脏干细胞 外泌体 磁珠分选 旁分泌效应 氧化应激 凋亡 

分 类 号：R332.11[医药卫生—人体生理学] R329.25[医药卫生—基础医学]