Sun X, Shi H, Zhang L, Liu Z, Li K, Qian L, Zhu X, Yang K, Fu Q, Ding H
Department of Orthopedics, Affiliated People's Hospital of Jiangsu University, Zhenjiang 212000, China.
Department of Orthopedics, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2024 Jan 20;44(1):119-128. doi: 10.12122/j.issn.1673-4254.2024.01.14.
To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells (EMSCs-exo) for repairing secondary spinal cord injury.
EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa, identified by transmission electron microscope, nanoparticle tracking analysis (NTA), and Western blotting, and quantified using the BCA method. Neonatal rat microglia purified by differential attachment were induced with 100 μg/L lipopolysaccharide (LPS) and treated with 37.5 or 75 mg/L EMSCs-exo. PC12 cells were exposed to 400 μmol/L HO and treated with EMSCs-exo at 37.5 or 75 mg/L. The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT- PCR, and the concentrations of IL- 6, IL-10, and IGF-1 in the supernatants were measured with ELISA. The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry.
The isolated rat EMSCs showed high expressions of nestin, CD44, CD105, and vimentin. The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63, CD81, and TSG101 but not vimentin. In LPS-treated microglia, EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression ( < 0.05). EMSCs-exo treatment at 75 mg/L, as compared with the lower concentration at 37.5 mg/L, more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia, and more effectively promoted cell survival and decreased apoptosis rate of HO-induced PC12 cells ( < 0.05).
EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival, suggesting its potential in controlling secondary spinal cord injury.
探讨大鼠外胚层间充质干细胞来源的外泌体（EMSCs-exo）对修复继发性脊髓损伤的潜在价值。
采用超速离心法从大鼠鼻黏膜分离的外胚层间充质干细胞中获取EMSCs-exo，通过透射电子显微镜、纳米颗粒跟踪分析（NTA）和蛋白质印迹法进行鉴定，并用BCA法进行定量。通过差速贴壁法纯化新生大鼠小胶质细胞，用100 μg/L脂多糖（LPS）诱导，并用37.5或75 mg/L的EMSCs-exo处理。将PC12细胞暴露于400 μmol/L HO中，并用37.5或75 mg/L的EMSCs-exo处理。用蛋白质印迹法和qRT-PCR测定处理后细胞中Arg1和iNOS的蛋白质和mRNA表达，用ELISA法测定上清液中IL-6、IL-10和IGF-1的浓度。用CCK-8法和流式细胞术检测PC12细胞的活力和凋亡情况。
分离的大鼠外胚层间充质干细胞高表达巢蛋白、CD44、CD105和波形蛋白。获得的EMSCs-exo在透射电子显微镜下具有典型的杯状结构，平均粒径为142 nm，CD63、CD81和TSG101呈阳性，波形蛋白呈阴性。在LPS处理的小胶质细胞中，75 mg/L的EMSCs-exo处理显著提高了Arg1蛋白水平，降低了iNOS蛋白表达（P<0.05）。与37.5 mg/L的较低浓度相比，75 mg/L的EMSCs-exo处理更强烈地增加了LPS诱导的小胶质细胞中Arg1 mRNA表达以及IGF-1和IL-10的产生，降低了iNOS mRNA表达和IL-6的产生，并且更有效地促进了HO诱导的PC12细胞的存活并降低了凋亡率（P<0.05）。
75 mg/L的EMSCs-exo可有效降低M1小胶质细胞比例，减轻氧化应激下的神经元凋亡，促进神经元存活，提示其在控制继发性脊髓损伤方面具有潜在作用。
