内皮祖细胞来源小细胞外囊泡促进小鼠脊髓损伤修复的实验研究

Experimental study of endothelial progenitor cells derived small extracellular vesicles for spinal cord injury repair in mice

目的探讨内皮祖细胞来源小细胞外囊泡(endothelial progenitor cells derived small extracellular vesicles,EPCs-sEVs)对小鼠脊髓损伤的治疗效果。方法取20只C57BL/6雄性小鼠股骨及胫骨骨髓分离培养EPCs,采用双荧光染色及流式细胞术鉴定;然后对EPCs进行传代,收集P2~P4代细胞上清,以超速离心法提取EPCs-sEVs,采用透射电镜、纳米流式检测仪及Western blot进行鉴定。取40只C57BL/6雌性小鼠随机分为4组(n=10),其中假手术组仅切除T10椎板,模型组以及低、高浓度干预组制备T10脊髓损伤模型;模型制备后30 min、3 d及7 d低、高浓度干预组经尾静脉分别注射浓度为1×109、1×1010个/mL的EPCs-sEVs。术后行小鼠行为学检测(BMS评分、斜板实验、Von Frey实验),术后4周取材行大体、HE染色及免疫组织化学染色观察脊髓组织结构变化。另取3只C57BL/6雌性小鼠制备T10脊髓损伤模型后,经尾静脉注入DiR标记的EPCs-sEVs,30 min后活体成像仪观察EPCs-sEVs是否达脊髓损伤部位。结果经鉴定,成功获取小鼠骨髓来源的EPCs及EPCssEVs;活体成像仪观察显示EPCs-sEVs在注射后30 min内募集至脊髓损伤区域。行为学检测示,术后2周内两干预组BMS评分及斜板实验最大角度与模型组比较差异均无统计学意义(P>0.05),2周后均明显优于模型组(P<0.05);术后4周两干预组Von Frey实验示机械痛阈值明显高于模型组、低于假手术组,差异均有统计学意义(P<0.05);上述指标两干预组间比较差异均无统计学意义(P>0.05)。与模型组相比,两干预组损伤节段脊髓组织缺损较小,单个核细胞浸润较少,组织结构恢复明显,血管新生更多(P<0.05);两干预组间无明显差异。结论 EPCs-sEVs可促进小鼠脊髓损伤修复,为脊髓损伤的生物治疗提供了新的方案。

Objective To explore the potential therapeutic effects of endothelial progenitor cells derived small extracellular vesicles(EPCs-sEVs) on spinal cord injury in mice. Methods 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(n=10). The mice were only removed T10 lamina in sham group, and prepared T10 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 1×109 and 1×1010 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 T10 spinal cord injury models, and DiR-labeled EPCssEVs were injected through the tail vein. After 30 minutes, in vivo imaging was used to observe whether the EPCs-sEVs reached the spinal cord injury site. Results After identification, EPCs and EPCs-sEVs derived from mouse bone marrow were successfully obtained. In vivo 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(P>0.05),while both were significantly better than the model group(P<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(P<0.05);there was no significant difference between two intervention groups(P>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(P<0.05);there was no significant difference between the two intervention groups.Conclusion 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.