集落形成内皮细胞--组织血管工程的候选培养物:基因和蛋白质组概况

M. Khanova, A. Kutikhin, V. Matveeva, E. Velikanova, E. O. Krivkina, L. Antonova
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引用次数: 0

摘要

目的通过与人脐静脉内皮细胞(HUVEC)和人冠状动脉内皮细胞(HCAEC)培养物的蛋白质组和基因表达谱的比较分析,验证 ECFC 培养物作为血管组织工程候选培养物的有效性。冠状动脉疾病患者的外周血单核细胞经培养后获得 ECFC 培养物。细胞用 TRIzol 裂解,用 Purelink RNA Micro Scale Kit 分离总 RNA,同时进行 DNase 处理。然后进行 rRNA 去库,接着创建 DNA 文库。在 CFX96 Touch Bio-Rad 放大器上使用定量聚合酶链反应对 DNA 文库进行量化。将 DNA 文库等摩尔混合,并在 HiSeq 2000(Illumina)上以 2x125 核苷酸的成对端读数进行测序。使用泛内皮标志物 CD31、vWF、VEG-FR2/KDR、内皮祖细胞标志物 CD34、上皮-间质转化标志物 Snail 和 Slug 以及内皮规范标志物:动脉 HEY2、静脉 COUP-TFII 和淋巴 LYVE1、VEGFR2 进行常规 Western 印迹。根据生产商的方案,使用 Proteome Profiler Human Angiogenesis Array Kit 对 55 种血管生成相关蛋白进行点印迹分析。ECFC过表达所有三种内皮系的标志物(与HCAEC相比,过表达KDR、VWF、CD34、NRP2、FLT4和LYVE1;与HUVEC相比,过表达NOTCH4、DLL2)和LYVE1。蛋白质组分析表明,就 HEY2、LYVE1、VEGFR3、Snail 和 Slug 的表达而言,ECFC 是介于 HCAEC 和 HU-VEC 之间的中间群体。在 ECFC 和 HUVEC 之间检测到 261 个 DEGs,在 ECFC 和 HCAEC 之间检测到 470 个 DEGs。内皮集落形成细胞的基因表达谱与成熟内皮细胞一致,表明 ECFC 是介于 HCAEC 和 HUVEC 之间的中间群体。建议将 ECFC 培养用于组织血管工程。
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Colony-forming endothelial cells – candidate culture for tissue vascular engineering: the gene and proteomic profile
Aim. To validate ECFC culture as a candidate culture for vascular tissue engineering using comparative analysis of the proteomic and gene expression profiles in comparison with cultures of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC).Materials and Methods. ECFC culture was obtained by cultivating peripheral blood mononuclear cells of patients with coronary artery disease. Commercial HCAECs produced by Cell Applications, and HUVECs cultured according to the modified protocol of Jaffe were used as controls.The cells were lysed with TRIzol, and total RNA was isolated using a Purelink RNA Micro Scale Kit with concomitant DNase treatment. Next, rRNA depletion was carried out, followed by the creation of DNA libraries. DNA libraries were quantified using quantitative polymerase chain reaction on a CFX96 Touch Bio-Rad amplifier. DNA libraries were equimolarly mixed and sequenced on HiSeq 2000 (Illumina) with a paired-end reads of 2x125 nucleotides.Conventional western blotting was performed using pan-endothelial markers CD31, vWF, VEG-FR2/KDR, marker of endothelial progenitor cells CD34, markers of epithelial-mesenchymal transition Snail and Slug, and markers of endothelial specification: arterial HEY2, venous COUP-TFII and lymphatic LYVE1, VEGFR2. Dot blotting against 55 angiogenesis-related proteins was performed using Proteome Profiler Human Angiogenesis Array Kit in accordance with the manufacturer's protocol.Results. ECFC overexpresses markers of all three endothelial lineages (KDR, VWF, CD34, NRP2, FLT4 and LYVE1 compared to HCAEC; NOTCH4, DLL2) and LYVE1 compared to HUVEC. Proteomic profiling indicated ECFC as an intermediate population between HCAEC and HU-VEC in term of the expression of HEY2, LYVE1, VEGFR3, Snail and Slug. 261 DEGs were detected between ECFC and HUVEC, and 470 DEGs between ECFC and HCAEC.Conclusion. The gene expression profile of endothelial colony-forming cells corresponds to mature endothelial cells and indicates ECFC as an intermediate population between HCAEC and HUVEC. ECFC culture can be recommended for tissue vascular engineering.
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