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首页> 美国卫生研究院文献>Cell Transplantation >Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model
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Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model

机译：通过质量和数量控制的培养富含内皮祖细胞的人外周血单核细胞在猪伤口模型中加速血管形成和伤口愈合

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The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.
机译：内皮祖细胞（EPC）的移植用于促进伤口血管生成。对于患有慢性伤口和伴发疾病的患者，EPC的数量和功能通常受到损害。为了克服这些限制，我们先前开发了一种质量和数量控制（QQ）培养系统，以丰富EPC中的外周血单个核细胞（PBMNC）。为了评估在QQ培养（QQMNC）后收获的单核细胞（MNC）的伤口愈合功效，对大型动物进行了临床前研究。在没有血管生成的细胞因子和生长因子存在下，在无血清培养基中培养从健康人类受试者的血液中收集的MNC 7天。将每个全层皮肤缺损或对照盐水总计5×10 6 QQMNC注射到环孢霉素免疫抑制的猪诱导的伤口中。 EPC菌落形成试验表明，QQMNC中有明显数量更多的确定的（部分分化的）EPC菌落形成单位。 QQMNC表面标记的流式细胞术评估显示CD34 + 和CD133 + 干细胞群富集，CCR2 + 细胞百分比显着降低，并且与PBMNCs相比，抗炎M2型巨噬细胞（CD206 + 细胞）的百分比增加了10倍以上。用QQMNC治疗的伤口闭合率明显更高。术后第21天收获伤口，冷冻并切片。苏木精和曙红染色显示，经QQMNC处理的伤口的上皮形成比对照组更先进。经过处理的伤口形成肉芽组织，胶原蛋白更成熟，毛细血管网络更大。 CD31和人类线粒体共染色证实了新生血管中存在分化的人类细胞。实时聚合酶链反应（PCR）显示伤口床中的白介素6（IL-6），IL-10和IL-4上调，表明所移植QQMNC的旁分泌活性。我们的数据首次证明，从少量外周血获得的MNC的QQ培养可产生对伤口愈合有效的血管生成和治疗细胞。

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