Wang Tao, Fang Xiao, Yin Zong-Sheng
Department of Orthopedics, the First Affiliated Hospital of Anhui Medical University; Department of Spine Surgery, Hefei Binhu Hospital, the Third Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China.
Department of Orthopedics, the First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China.
Neural Regen Res. 2018 May;13(5):887-895. doi: 10.4103/1673-5374.232484.
Endothelial progenitor cells secrete a variety of growth factors that inhibit inflammation, promote angiogenesis and exert neuroprotective effects. Therefore, in this study, we investigated whether endothelial progenitor cell-conditioned medium might have therapeutic effectiveness for the treatment of spinal cord injury using both in vitro and in vivo experiments. After primary culture of bone marrow-derived macrophages, lipopolysaccharide stimulation was used to classically activate macrophages to their proinflammatory phenotype. These cells were then treated with endothelial progenitor cell-conditioned medium or control medium. Polymerase chain reaction was used to determine mRNA expression levels of related inflammatory factors. Afterwards, primary cultures of rat spinal cord neuronal cells were prepared and treated with HO and either endothelial progenitor cell-conditioned medium or control medium. Hoechst 33258 and propidium iodide staining were used to calculate the proportion of neurons undergoing apoptosis. Aortic ring assay was performed to assess the effect of endothelial progenitor cell-conditioned medium on angiogenesis. Compared with control medium, endothelial progenitor cell-conditioned medium mitigated the macrophage inflammatory response at the spinal cord injury site, suppressed apoptosis, and promoted angiogenesis. Next, we used a rat model of spinal cord injury to examine the effects of the endothelial progenitor cell-conditioned medium in vivo. The rats were randomly administered intraperitoneal injection of PBS, control medium or endothelial progenitor cell-conditioned medium, once a day, for 6 consecutive weeks. Immunohistochemistry was used to observe neuronal morphology. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was performed to detect the proportion of apoptotic neurons in the gray matter. The Basso, Beattie and Bresnahan Locomotor Rating Scale was used to evaluate the recovery of motor function of the bilateral hind limbs after spinal cord injury. Compared with the other two groups, the number of axons was increased, cavities in the spinal cord were decreased, the proportion of apoptotic neurons in the gray matter was reduced, and the Basso, Beattie and Bresnahan score was higher in the endothelial progenitor cell-conditioned medium group. Taken together, the in vivo and in vitro results suggest that endothelial progenitor cell-conditioned medium suppresses inflammation, promotes angiogenesis, provides neuroprotection, and promotes functional recovery after spinal cord injury.
内皮祖细胞分泌多种生长因子，这些因子可抑制炎症、促进血管生成并发挥神经保护作用。因此，在本研究中，我们通过体外和体内实验研究了内皮祖细胞条件培养基对脊髓损伤治疗是否具有疗效。在对骨髓来源的巨噬细胞进行原代培养后，使用脂多糖刺激将巨噬细胞经典激活为促炎表型。然后用内皮祖细胞条件培养基或对照培养基处理这些细胞。采用聚合酶链反应测定相关炎症因子的mRNA表达水平。之后，制备大鼠脊髓神经元细胞原代培养物，并用HO以及内皮祖细胞条件培养基或对照培养基进行处理。使用Hoechst 33258和碘化丙啶染色计算凋亡神经元的比例。进行主动脉环试验以评估内皮祖细胞条件培养基对血管生成的影响。与对照培养基相比，内皮祖细胞条件培养基减轻了脊髓损伤部位的巨噬细胞炎症反应，抑制了细胞凋亡，并促进了血管生成。接下来，我们使用大鼠脊髓损伤模型在体内研究内皮祖细胞条件培养基的作用。大鼠被随机腹腔注射PBS、对照培养基或内皮祖细胞条件培养基，每天一次，连续6周。采用免疫组织化学观察神经元形态。进行末端脱氧核苷酸转移酶介导的dUTP缺口末端标记试验以检测灰质中凋亡神经元的比例。使用Basso、Beattie和Bresnahan运动评分量表评估脊髓损伤后双侧后肢运动功能的恢复情况。与其他两组相比，内皮祖细胞条件培养基组的轴突数量增加，脊髓空洞减少，灰质中凋亡神经元的比例降低，Basso、Beattie和Bresnahan评分更高。综上所述，体内和体外结果表明，内皮祖细胞条件培养基可抑制炎症、促进血管生成、提供神经保护并促进脊髓损伤后的功能恢复。
