Hu Cheng-heng, Li Zhi-ming, DU Zhi-min, Zhang Ai-xia, Yang Da-ya, Wu Gui-fu
Ministry of Health of China, Sun Yat-sen University, Guangzhou, Guangdong, China.
Chin Med J (Engl). 2009 Mar 5;122(5):548-55.
Cell-based vascular therapies of endothelial progenitor cells (EPCs) mediated neovascularization is still a novel but promising approach for the treatment of ischemic disease. The present study was designed to investigate the therapeutic potentials of human umbilical cord blood-derived EPCs (hUCB-EPCs) in rat with acute myocardial infarction.
Human umbilical cord blood (hUCB) mononuclear cells were isolated using density gradient centrifugation from the fresh human umbilical cord in healthy delivery woman, and cultured in M199 medium for 7 days. The EPCs were identified by double-positive staining with 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine percholorate-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and fluorescein isothiocyanate-conjugated Ulex europaeus lectin (FITC-UEA-l). The rat acute myocardial infarction model was established by the ligation of the left anterior descending artery. The hUCB-EPCs were intramyocardially injected into the peri-infarct area. Four weeks later, left ventricular function was assessed by a pressure-volume catheter. The average capillary density (CAD) was evaluated by anti-VIII immunohistochemistry staining to reflect the development of neovascularization at the peri-infarct area. The graft cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibody, representing human origin of EPCs and vascular endothelium, respectively. Expressions of cytokines, proliferating cell nuclear angigen (PCNA), platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor (VEGF) were detected to investigate the underlying mechanisms of cell differentiation and revascularization.
The donor EPCs were detectable and integrated into the host myocardium as confirmed by double-positive immunofluorescence staining with HNA and CD31. And the anti-VIII staining demonstrated a higher degree of microvessel formation in EPCs transplanted rats, associated with a significant improvement of global heart function in terms of the increase of left ventricular end-systolic pressure (LVESP), +dp/dtmax and -dp/dtmax as well as the decrease of LVEDP in rats with EPCs therapy comparing to the control rats (P < 0.05). Moreover, the expression of the rat PCNA mRNA and PECAM were both enhanced in the EPCs group compared with that of the control group.
The human umbilical cord blood-derived EPCs could incorporate into new-born capillaries in rat myocardium, induce revascularization and improve the proliferation activity in the peri-infarct area, resulting in the improvement of global heart function. This may indicate a promising stem cell resource in cell-based therapy for ischaemic diseases.
基于内皮祖细胞（EPCs）的细胞血管疗法介导的新生血管形成仍是一种治疗缺血性疾病的新型且有前景的方法。本研究旨在探讨人脐带血来源的EPCs（hUCB - EPCs）对急性心肌梗死大鼠的治疗潜力。
从健康产妇的新鲜脐带中通过密度梯度离心法分离人脐带血（hUCB）单个核细胞，并在M199培养基中培养7天。通过用1,1'-二油酰基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐标记的乙酰化低密度脂蛋白（Dil - Ac - LDL）和异硫氰酸荧光素偶联的荆豆凝集素（FITC - UEA - 1）进行双阳性染色来鉴定EPCs。通过结扎左冠状动脉前降支建立大鼠急性心肌梗死模型。将hUCB - EPCs心肌内注射到梗死周边区域。四周后，用压力 - 容积导管评估左心室功能。通过抗VIII免疫组织化学染色评估平均毛细血管密度（CAD），以反映梗死周边区域新生血管形成的情况。通过用人核抗原（HNA）和CD31抗体进行双免疫荧光染色来鉴定移植细胞，分别代表EPCs的人源性质和血管内皮。检测细胞因子、增殖细胞核抗原（PCNA）、血小板内皮细胞黏附分子（PECAM）和血管内皮生长因子（VEGF）的表达，以研究细胞分化和血管再生的潜在机制。
通过HNA和CD31双阳性免疫荧光染色证实供体EPCs可被检测到并整合到宿主心肌中。并且抗VIII染色显示EPCs移植大鼠的微血管形成程度更高，与对照大鼠相比，EPCs治疗组大鼠的左心室收缩末期压力（LVESP）、 +dp/dtmax和 -dp/dtmax增加以及左心室舒张末期压力（LVEDP）降低，整体心脏功能有显著改善（P < 0.05）。此外，与对照组相比，EPCs组大鼠的PCNA mRNA和PECAM表达均增强。
人脐带血来源的EPCs可整合到大鼠心肌的新生毛细血管中，诱导血管再生并改善梗死周边区域的增殖活性，从而改善整体心脏功能。这可能表明在基于细胞的缺血性疾病治疗中，hUCB - EPCs是一种有前景的干细胞资源。
