Lin Junqing, Huang Tengli, Gao Tao, Zheng Xianyou
Department of Orthopedic Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, 200233, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2021 Apr 15;35(4):488-495. doi: 10.7507/1002-1892.202009130.
To explore the potential therapeutic effects of endothelial progenitor cells derived small extracellular vesicles (EPCs-sEVs) on spinal cord injury in mice.
EPCs were separated from femur and tibia bone marrow of 20 C57BL/6 male mice, and identified by double fluorescence staining and flow cytometry. Then the EPCs were passaged and the cell supernatants from P2-P4 generations EPCs were collected; the EPCs-sEVs were extracted by ultracentrifugation and identified by transmission electron microscopy, nanoflow cytometry, and Western blot. Forty C57BL/6 female mice were randomly divided into 4 groups ( =10). The mice were only removed T lamina in sham group, and prepared T spinal cord injury models in the model group and the low and high concentration intervention groups. After 30 minutes, 3 days, and 7 days of operation, the mice in low and high concentration intervention groups were injected with EPCs-sEVs at concentrations of 1×10 and 1×10 cells/mL through the tail vein, respectively. The behavioral examinations [Basso Mouse Scale (BMS) score, inclined plate test, Von Frey test] , and the gross, HE staining, and immunohistochemical staining were performed to observe the structural changes of the spinal cord at 4 weeks after operation. Another 3 C57BL/6 female mice were taken to prepare T spinal cord injury models, and DiR-labeled EPCs- sEVs were injected through the tail vein. After 30 minutes, imaging was used to observe whether the EPCs-sEVs reached the spinal cord injury site.
After identification, EPCs and EPCs-sEVs derived from mouse bone marrow were successfully obtained. imaging of the spinal cord showed that EPCs-sEVs were recruited to the spinal cord injury site within 30 minutes after injection. There was no significant difference in BMS scores and the maximum angle of the inclined plate test between two intervention groups and the model group within 2 weeks after operation ( >0.05), while both were significantly better than the model group ( <0.05) after 2 weeks. The Von Frey test showed that the mechanical pain threshold of the two intervention groups were significantly higher than that of model group and lower than that of sham group ( <0.05); there was no significant difference between two intervention groups ( >0.05). Compared with the model group, the injured segment of the two intervention groups had smaller spinal cord tissue defects, less mononuclear cells infiltration, more obvious tissue structure recovery, and more angiogenesis, and these differences were significant ( <0.05); there was no significant difference between the two intervention groups.
EPCs-sEVs can promote the repair of spinal cord injury in mice and provide a new plan for the biological treatment of spinal cord injury.
探讨内皮祖细胞来源的小细胞外囊泡（EPCs-sEVs）对小鼠脊髓损伤的潜在治疗作用。
从20只C57BL/6雄性小鼠的股骨和胫骨骨髓中分离出EPCs，并通过双荧光染色和流式细胞术进行鉴定。然后对EPCs进行传代培养，收集第2-4代EPCs的细胞上清液；通过超速离心提取EPCs-sEVs，并通过透射电子显微镜、纳米流式细胞术和蛋白质免疫印迹法进行鉴定。将40只C57BL/6雌性小鼠随机分为4组（每组n = 10）。假手术组仅切除T椎板，模型组和低、高浓度干预组制备T脊髓损伤模型。术后30分钟、3天和7天，低、高浓度干预组的小鼠分别通过尾静脉注射浓度为1×10和1×10个细胞/mL的EPCs-sEVs。进行行为学检测[巴索小鼠评分（BMS）、斜板试验、von Frey试验]，并在术后4周进行大体观察、苏木精-伊红（HE）染色和免疫组织化学染色，以观察脊髓的结构变化。另取3只C57BL/6雌性小鼠制备T脊髓损伤模型，并通过尾静脉注射DiR标记的EPCs-sEVs。30分钟后，采用成像技术观察EPCs-sEVs是否到达脊髓损伤部位。
经鉴定，成功获得了源自小鼠骨髓的EPCs和EPCs-sEVs。脊髓成像显示，EPCs-sEVs在注射后3分钟内被募集到脊髓损伤部位。术后2周内，两个干预组与模型组的BMS评分和斜板试验最大角度无显著差异（P>0.05），而术后2周后均显著优于模型组（P<0.05）。von Frey试验显示，两个干预组的机械性疼痛阈值均显著高于模型组且低于假手术组（P<0.05）；两个干预组之间无显著差异（P>0.05）。与模型组相比，两个干预组损伤节段的脊髓组织缺损较小，单核细胞浸润较少，组织结构恢复更明显，血管生成更多，且这些差异具有统计学意义（P<0.05）；两个干预组之间无显著差异。
EPCs-sEVs可促进小鼠脊髓损伤的修复，为脊髓损伤的生物治疗提供了新的方案。
