骨髓间充质干细胞源外泌体调控心肌微血管内皮细胞增殖的机制研究

Mechanism of exosomes released from bone marrow stem cells regulating proliferation of cardiac microvascular endothelial cells

目的探讨缺氧预处理骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源的外泌体(exosomes)调控心肌微血管内皮细胞(cardiac microvascular endothelial cells,CMVECs)增殖的机制。方法采用酶消化法联合差速贴壁法培养大鼠CMVECs,超高速离心法提取常氧及缺氧预处理的BMSCs exosomes,免疫荧光检测CMVECs内化exosomes情况。实验分为4组(n=3):①正常对照组(Control),②去除exosomes后的BMSCs条件培养基的空白对照组(Free-Exos),③常氧外泌体组(Nor-Exos),④缺氧外泌体组(Hypoxia-Exos,外泌体浓度为400μg/μL,与CMVECs共培养24 h)。采用Edu法及流式细胞术细胞周期法检测细胞增殖能力,qRT-PCR检测exosomes和CMVECs各组中miR-214表达变化。抑制exosomes中miR-214后,qRT-PCR检测exosomes及CMVECs中miR-214的表达变化,并观察细胞增殖情况。结果经透射电镜及免疫印迹法验证提取的囊泡状物质为外泌体,并通过免疫荧光分析发现DiI标记的exosomes可被CMVECs内化;Edu及细胞周期结果显示,Hypoxia-Exos组较Nor-Exos组具有更强的促CMVECs增殖能力(P<0.05)。qRT-PCR结果显示,与Nor-Exos组比较,Hypoxic-Exos组中miR-214表达显著升高(P<0.05)。下调Hypoxia-Exos中的miR-214表达后,Edu及细胞周期结果显示Hypoxia-Exos组的促进细胞增殖能力被部分阻断(P<0.05)。结论缺氧预处理骨髓间充质干细胞源exosomes可能通过传递miR-214促进CMVECs增殖效应。

ObjectiveTo investigate the mechanism of exosomes released from bone marrow stem cells(BMSCs)on the proliferation in cardiac microvascular endothelial cells(CMVECs)by miR-214 regulation.MethodsRat CMVECs were cultured by enzyme digestion combined with differential adherence.Ultra-high speed centrifugation was used to extract exosomes from normal-oxygen or hypoxic-pretreated BMSCs.Immunofluorescence assay was employed to track the internalization of exosomes in CMVECs.Then the BMSCs were divided into 4 groups:①control group,②free exosomes(Free-Exos),③normal exosomes(Nor-Exos),and④hypoxia exosomes(Hypoxia-Exos,400μg/μL exosome co-cultured with CMVECs for 24 h).The cell proliferation was measured by Edu assay and flow cytometry.The miR-214 levels in exosomes or CMVECs were assessed using qRT-PCR,and the levels were measured again after miR-214 in exosomes were inhibited.And the cell proliferation was observed again by the above methods.Finally,after liposome 2000 was employed to transfect miR-214 mimics into CMVECs,the cell proliferation ability was measured by the Edu assay.ResultsTransmission electron microscopy and immunoblotting indicated that the extracted vesicles were exosomes.Immunofluorescence assay revealed that DiI-labeled exosomes could be internalized by CMVECs.Edu assay and cell cycle assay showed that Hypoxic-Exos had stronger potential to promote cell proliferation than Nor-Exos in CMVECs.qRT-PCR results showed that miR-214 was highly expressed in Hypoxic-Exos compared with Nor-Exos(P<0.05).In CMVECs,the expression of miR-214 in Hypoxia-Exos group was significantly higher than that in Control group and Nor-Exos group(P<0.05).In addition,Hypoxia-Exos’s ability to promote cell proliferation was partially blocked by down-regulation of miR-214 in Hypoxia-Exos.However,after overexpression of mir-214 in CMVECs,cell proliferation ability was enhanced.ConclusionExosomes released from hypoxic-pretreated BMSCs can promote CMVECs proliferation by medicating miR-214.