Kong Zhaohong, Chen Meixin, Jiang Jian, Zhu Jiang, Liu Yumin
Department of Neurology, Renmin Hospital of Wuhan University, Wuhan, China.
Department of Neurology, Zhongnan Hospital of Wuhan University, Wuhan, China.
Cardiovasc Diagn Ther. 2020 Oct;10(5):1270-1279. doi: 10.21037/cdt-20-536.
Endothelial progenitor cells (EPCs) play an important role in the re-endothelialization of ischemic cerebrovascular disease. However, the current acquisition method has some deficiencies. This study aimed to design a new and practical method for obtaining EPCs.
Bone marrow was obtained autologously from the right tibia of living rats. Briefly, the right tibia bone was carefully exposed and two holes (1 mm in diameter) were made in the tuberosity and lower one-third of the tibia, respectively. A PE-50 catheter and syringe (5 mL) were inserted through the holes to aspirate the bone marrow. Bone marrow mononuclear cells (BMMCs) were isolated by density-gradient centrifugation with Ficoll and counted. Adherent cell culture continued for 2 weeks, and the medium was replaced every 3 days.
During the first days of culture, adherent cells formed a monolayer, consisting predominantly of small-sized cells. Single large cells with endothelial morphology were observed. On day 4, the nonadherent cells were removed, and the adherent cells were left for further culture. On day 6-7, a proliferating population of round cells formed clusters in the culture chamber, and morphological analysis revealed a homogeneous population of colony-forming units (CFUs). Large, flat cells with endothelial morphology sprouted from the CFUs, which had nearly disappeared by day 14 of culture. The adherent cells were positive for CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), internalized acetylated low-density lipoprotein, and bound ulex europaeus-agglutinin-I, but were negative for CD45, which correlated with the endothelial morphology and ability to form capillaries of EPCs.
Our results are direct evidence that mononuclear cells (MCS) from living rat bone marrow can be used to culture EPCs under certain culture conditions, providing a new method for the further study of autologous EPC transplantation.
内皮祖细胞（EPCs）在缺血性脑血管疾病的再内皮化过程中发挥着重要作用。然而，目前的获取方法存在一些不足之处。本研究旨在设计一种新的实用方法来获取EPCs。
从活体大鼠右胫骨自体获取骨髓。简要地说，小心暴露右胫骨，分别在胫骨结节和下三分之一处制作两个直径为1mm的孔。将PE - 50导管和5mL注射器通过孔插入以抽吸骨髓。通过Ficoll密度梯度离心分离骨髓单个核细胞（BMMCs）并计数。贴壁细胞培养持续2周，每3天更换培养基。
在培养的最初几天，贴壁细胞形成单层，主要由小细胞组成。观察到具有内皮形态的单个大细胞。在第4天，去除非贴壁细胞，留下贴壁细胞进行进一步培养。在第6 - 7天，一群增殖的圆形细胞在培养室中形成簇，形态学分析显示为均匀的集落形成单位（CFUs）群体。具有内皮形态的大的扁平细胞从CFUs中长出，在培养第14天时CFUs几乎消失。贴壁细胞CD133和血管内皮生长因子受体2（VEGFR2）呈阳性，内化乙酰化低密度脂蛋白，并结合荆豆凝集素 - I，但CD45呈阴性，这与EPCs的内皮形态和形成毛细血管的能力相关。
我们的结果直接证明，在一定培养条件下，活体大鼠骨髓中的单个核细胞（MCS）可用于培养EPCs，为自体EPC移植的进一步研究提供了一种新方法。
