中性鞘磷脂酶2对缺氧预处理间充质干细胞来源的外泌体生物学特性的影响

Effects of neutral sphingomyelinase 2 on biological characteristics of exosomes derived from mesenchymal stem cells under hypoxia precondition

目的探讨中性鞘磷脂酶2对缺氧预处理骨髓间充质干细胞来源的外泌体生物学特性的影响。方法分离并体外扩增6周龄C57BL/6小鼠来源的骨髓间充质干细胞,收集细胞上清,分离来自缺氧处理的外泌体(ExoH)和常氧下培养来源的外泌体(ExoN),并鉴定其生物学特性。比较ExoH和ExoN在体内实验中对心肌细胞保护作用的差异,同时收集在缺氧预处理状态下加入中性鞘磷脂酶2抑制剂GW4869来源的外泌体(ExoH+GW),并在体外实验中比较ExoN、ExoH和ExoH+GW对心肌细胞抗凋亡及血管新生能力的作用。结果ExoH组小鼠心肌梗死后28 d的射血分数显著高于ExoN组(44.12%±8.12%比20.73%±7.32%,n=20,t=11.95,P<0.05)。同时发现,缺氧增加中性鞘磷脂酶2的表达,而ExoH+GW组在体外管腔形成及心肌细胞保护方面明显弱于ExoH组,并且其微小RNA-210含量显著减低。结论缺氧预处理骨髓间充质干细胞来源的外泌体拥有更好的心脏保护能力,中性鞘磷脂酶2可能是主要的作用机制,可能通过影响外泌体内有益微小RNA的含量来发挥生物学作用。

Objective To investigate neutral sphingomyelinase 2(nSMase2)on biological effects of exosomes derived from mesenchymal stem cells(MSCs)under hypoxia precondition.Methods MSCs bone marrow from C57BL/66-week mouse were isolated and cultured,and surem was collected.Exosomes from MSCs under normoxia(ExoN)and hypoxia(ExoH)were isolated and their biological characteristcs were identified.The capacity of cardiac protection in vivo was compared between ExoH and ExoN.Then exosome(ExoH+GW)derived from the nSMase2 inhibitor GW4869 added under hypoxic precondition was isolated.The in vitro anti-apoptosis and angiogenesis capacity were compared among ExoN,ExoH and ExoH+GW groups.Results The ejection fraction of ExoH group was significantly higher than that of ExoN group at 28 days after myocardial infarction(44.12%±8.12%vs.20.73±7.32%,n=20,t=11.95,P<0.05).And expression of nSMase2 increased under hypoxic condition,while tube-formation and cardiomyocytes protection assay was significantly weaker in ExoH+GW group than that in ExoH group in vitro,and the miR-210 content significantly decreased in ExoH+GW group.Conclusions Hypoxic pretreatment of bone marrow mesenchymal stem cell-derived exosomes has better cardioprotective ability.nSMase2 may play a key biologic role by affecting the beneficial microRNA content in exosomes.