The aim of this study was to determine whether the combination of MSC implantation with miRNA-126-3p overexpression would further improve the surgical results after vein grafting. human umbilical cord MSCs (hucMSCs) and human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and characterized by a series of experiments. Lentivirus vector encoding miRNA-126-3p was transfected into hucMSCs and verified by PCR. We analyzed the miRNA-126-3p-hucMSC function in vascular endothelial cells by using a series of co-culture experiments. miRNA-126-3p-hucMSCs-exosomes were separated from cell culture supernatants and identified by WB and TEM. We validated the role of miRNA-126-3p-hucMSCs-exosomes on HUVECs proliferative and migratory and angiogenic activities by using a series of function experiments. We further performed co-culture experiments to detect downstream target genes and signaling pathways of miRNA-126-3p-hucMSCs in HUVECs. We established a rat vein grafting model, CM-Dil-labeled hucMSCs were injected intravenously into rats, and the transplanted cells homing to the vein grafts were detected by fluorescent microscopy. We performed historical and immunohistochemical experiments to exam miRNA-126-3p-hucMSC transplantation on vein graft neointimal formation and reendothelialization in vitro. We successfully isolated and identified primary hucMSCs and HUVECs. Primary hucMSCs were transfected with lentiviral vectors carrying miRNA-126-3p at a MOI 75. Co-culture studies indicated that overexpression of miRNA-126-3p in hucMSCs enhanced HUVECs proliferation, migration, and tube formation in vivo. We successfully separated hucMSCs-exosomes and found that miRNA-126-3p-hucMSCs-exosomes can strengthen the proliferative, migratory, and tube formation capacities of HUVECs. Further PCR and WB analysis indicated that, SPRED-1/PIK3R2/AKT/ERK1/2 pathways are involved in this process. In the rat vein arterialization model, reendothelialization analysis showed that transplantation with hucMSCs modified with miRNA-126-3p had a higher reendothelialization of the vein grafts. The subsequent historical and immunohistochemical examination revealed that delivery with miRNA-126-3p overexpressed hucMSCs significantly reduced vein graft intimal hyperplasia in rats. These results suggest hucMSC-based miRNA-126-3p gene therapy may be a novel option for the treatment of vein graft disease after CABG.

中文翻译：

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人脐带间充质干细胞携带的 miRNA-126-3p 通过体外外泌体介导的机制增强内皮功能并减弱体内静脉移植新内膜的形成

本研究的目的是确定MSC植入与miRNA-126-3p过表达的组合是否会进一步改善静脉移植后的手术效果。从人脐带中分离出人脐带间充质干细胞（hucMSCs）和人脐静脉内皮细胞（HUVECs），并通过一系列实验对其进行表征。将编码 miRNA-126-3p 的慢病毒载体转染到 hucMSCs 中并通过 PCR 验证。我们通过一系列共培养实验分析了血管内皮细胞中 miRNA-126-3p-hucMSC 的功能。将 miRNA-126-3p-hucMSCs-外泌体从细胞培养上清液中分离出来，并通过 WB 和 TEM 进行鉴定。我们通过一系列功能实验验证了 miRNA-126-3p-hucMSCs-外泌体对 HUVECs 增殖、迁移和血管生成活动的作用。我们进一步进行了共培养实验，以检测 HUVECs 中 miRNA-126-3p-hucMSCs 的下游靶基因和信号通路。我们建立了大鼠静脉移植模型，将CM-Dil标记的hucMSCs静脉注射到大鼠体内，并通过荧光显微镜检测移植细胞归巢到静脉移植物。我们进行了历史和免疫组织化学实验，以检查 miRNA-126-3p-hucMSC 移植对静脉移植新内膜形成和体外再内皮化的影响。我们成功地分离和鉴定了原发性 hucMSCs 和 HUVECs。以 MOI 75 携带 miRNA-126-3p 的慢病毒载体转染原代 hucMSCs。共培养研究表明，在 hucMSCs 中过表达 miRNA-126-3p 可增强 HUVECs 在体内的增殖、迁移和管形成。我们成功分离了hucMSCs-外泌体，发现miRNA-126-3p-hucMSCs-外泌体可以增强HUVECs的增殖、迁移和管形成能力。进一步的PCR和WB分析表明，SPRED-1/PIK3R2/AKT/ERK1/2通路参与了这一过程。在大鼠静脉动脉化模型中，再内皮化分析表明，移植 miRNA-126-3p 修饰的 hucMSCs 具有更高的静脉移植物再内皮化。随后的历史和免疫组织化学检查显示，用 miRNA-126-3p 过表达的 hucMSCs 递送显着减少了大鼠静脉移植物内膜增生。这些结果表明，基于 hucMSC 的 miRNA-126-3p 基因治疗可能是 CABG 后静脉移植疾病治疗的新选择。