Department of Anatomy, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, China.
Department of Neurosurgery, Affiliated Baiyun Hospital, Guizhou Medical University, Guiyang, China.
Cell Reprogram. 2021 Feb;23(1):47-56. doi: 10.1089/cell.2020.0055. Epub 2021 Jan 5.
Edaravone can induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into neuron-like cells and replace lost cells by transplanting neuron-like cells to repair spinal cord injury (SCI). In this study, BMSCs were derived from the bone marrow of male Wistar rats (4 weeks old) through density gradient centrifugation (1.073 /mL), and the cell purity of BMSCs was up to 95%. The combined injection of basic fibroblast growth factor and edaravone was conducted to differentiate BMSCs into neuron-like cells. In this study, 120 male Wistar rats were used to establish the model of semitransverse SCI; on the seventh day, neuron-like cells were labeled by BrdU and then injected into the epicenter of the injury of rats. On the 14th day after cell transplantation, the biotin dextran amine (BDA) fluorescent agent was used to track the repair of nerve damage. At 7, 14, 21, and 30 days after SCI, the Basso, Beattie, and Bresnahan (BBB) locomotor scale method was used to measure the functional recovery of hind limbs in rats. Additionally, hematoxylin and eosin (H&E) staining, Nissl staining, immunohistochemistry, transmission electron microscopy (TEM), Western blotting, and Real-time quantitative reverse transcripion PCR (qRT-PCR) were used to observe the regeneration of nerve cells. In the edaravone+BMSC group, behavioral analysis of locomotor function showed that functional recovery was significantly enhanced after transplantation of the cells, BrdU-positive cells could be observed scattered in the injured area and extended to both the head and tail, and the BDA tracer shows that the edaravone+BMSC group emits more fluorescent signals. Additionally, H&E staining, Nissl staining, and immunohistochemistry revealed that the space of spinal cord tissue was attenuated and the neurons were increased. Western blotting and qRT-PCR showed that the expression levels of neuron-specific enolase (NSE), Nestin, and neurofilament 200 (NF) were increased, while the expression of glial fibrillary acidic protein (GFAP) was decreased. TEM showed that cytoplasmic edema was reduced, mitochondrial vacuoles were attenuated, and nuclear chromatin concentration was declined after transplantation of neuron-like cells. Moreover, with the extension of time of edaravone+BMSC transplantation, the structures of mitochondria and endoplasmic reticulum tended to be normal. In summary, the induced differentiation of BMSC transplantation can significantly promote the functional repair of SCI.
依达拉奉可通过诱导骨髓间充质干细胞(BMSCs)分化为神经元样细胞,并通过移植神经元样细胞来修复脊髓损伤(SCI),从而替代受损细胞。在本研究中,通过密度梯度离心(1.073/mL)从雄性 Wistar 大鼠(4 周龄)的骨髓中分离出 BMSCs,BMSCs 的细胞纯度可达 95%。通过碱性成纤维细胞生长因子和依达拉奉联合注射诱导 BMSCs 分化为神经元样细胞。本研究采用 120 只雄性 Wistar 大鼠建立半横断 SCI 模型;在细胞移植后第 7 天,通过 BrdU 标记神经元样细胞,然后将其注入大鼠损伤中心。在细胞移植后第 14 天,使用生物素葡聚糖胺(BDA)荧光剂追踪神经损伤的修复情况。在 SCI 后 7、14、21 和 30 天,采用 Basso、Beattie 和 Bresnahan(BBB)运动评分法测量大鼠后肢的功能恢复情况。此外,还进行了苏木精和伊红(H&E)染色、尼氏染色、免疫组织化学、透射电子显微镜(TEM)、Western blot 和实时定量逆转录 PCR(qRT-PCR),以观察神经细胞的再生情况。在依达拉奉+BMSC 组中,运动功能行为分析显示细胞移植后功能恢复明显增强,可观察到 BrdU 阳性细胞散在分布于损伤区,并向头尾延伸,BDA 示踪剂显示依达拉奉+BMSC 组发出更多荧光信号。此外,H&E 染色、尼氏染色和免疫组织化学显示脊髓组织的空间减弱,神经元增多。Western blot 和 qRT-PCR 显示神经元特异性烯醇化酶(NSE)、巢蛋白和神经丝 200(NF)的表达水平增加,而神经胶质纤维酸性蛋白(GFAP)的表达水平降低。TEM 显示移植神经元样细胞后细胞质水肿减轻,线粒体空泡减轻,核染色质浓度降低。此外,随着依达拉奉+BMSC 移植时间的延长,线粒体和内质网的结构趋于正常。综上所述,BMSC 移植的诱导分化可显著促进 SCI 的功能修复。