标题：小鼠创伤性脑损伤后血清外泌体粒径和miR-21-5P表达水平的变化及意义

      作者：支敏 1，2，江孟晓 3，童有华 3，郑旭东 1，支慧 3    支敏 1，2，江孟晓 3，童有华 3，郑旭东 1，支慧 31.郑州大学基础医学院，河南 郑州 450066；2.郑州市人民检察院，河南 郑州 450846；3.皖南医学院病理解剖教研室，安徽 芜湖 241002

      卷次： 2023年34卷19期

      【摘要】 目的 观察轻型创伤性脑损伤(mTBI)小鼠血清外泌体物理特征与外泌体miR-21-5P的表达水平，并分析外泌体的粒径及miR-21-5P表达对脑外伤诊断的价值。方法 选择 8周龄C57小鼠 12只，按随机数表法分为mTBI组和假手术组(sham组)各 6只；采用 Feency法制作mTBI小鼠模型。第 1天、第 7天、第 21天后取小鼠外周血，第 3天、第 7天、第 14天、第 28天后对小鼠进行神经功能缺失评分(mNSS)。采用 PEG沉淀法提取外周血外泌体，运用纳米颗粒视踪技术(NTA)分析外泌体，采用定量 PCR检测外泌体中miR-21-5P的表达水平。结果 mTBI损伤组小鼠外周血外泌体在损伤发生1 d后其生成量为(0.84±0.24)×107 particals/mL，明显低于假手术组的(10.83±0.7)×107 particals/mL，且在损伤发生第28天mTBI损伤组小鼠外周血的外泌体虽然有所增加，但依然明显低于假手术组，差异均具有统计学意义(P<0.05)；同时，外泌体粒径检测结果显示，mTBI发生后的第一天，mTBI组小鼠外周血外泌体的粒径为(131.1±2.27) nm，明显多于 sham组小鼠的(114.6±6.01) nm，差异具有统计学意义(P<0.05)，且直到损伤第28天时，mTBI组小鼠外周血外泌体的粒径依然明显大于 sham组，差异均具有统计学意义(P<0.05)。定量PCR检测结果表明，mTBI组小鼠外周血外泌体中的miR-21-5P表达水平不仅在损伤24 h明显高于 sham组，在损伤发生后第 28天依然维持在较高水平，明显高于 sham组小鼠，差异均具有统计学意义(P<0.05)。结论 创伤性脑损伤后外周血外泌体不仅生成量与粒径均有明显变化，其所携带的miR-21-5P表达水平亦明显升高，且这些变化在损伤发生28 d时依然存在。可见，外周血外泌体的生成量、粒径及其携带的miR-21-5P对mTBI损伤的临床评估具有较高的价值，有望成为mTBI预后评估的潜在生物指标。
      【关键词】 小鼠；轻型创伤性脑损伤；外周血外泌体；外泌体生成量；外泌体粒径；miR-21-5P
      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2023）19—2737—06

Changes and significance of plasma exosome particle size and miR-21-5P expression levels after traumatic braininjury in mice.
ZHI Min 1,2, JIANG Meng-xiao 3, TONG You-hua 3, ZHENG Xu-dong 1, ZHI Hui 3. 1. School of BasicMedicine, Zhengzhou University, Zhengzhou 450066, Henan, CHINA; 2. Zhengzhou People's Procuratorate of HenanProvince, Zhengzhou 450846, Henan, CHINA; 3. Department of Pathology, Wannan Medical College, Wuhu 241002, Anhui,CHINA
【Abstract】 Objective To investigate the physical characteristics of serum exosomes and the expression levelof miR-21-5P in mice with mild traumatic brain injury (mTBI), and analyze the value of exosome particle size andmiR-21-5P expression in the diagnosis of brain injury. Methods Twelve 8-week-old C57 mice were randomly dividedinto mTBI group and sham group, with 6 mice in each group. mTBI mouse models were established using Feency meth-od. On the 1st, 7th, and 21st days, peripheral blood was collected from mice, and on the 3rd, 7th, 14th, and 28th days, neurolog-ical deficit score (mNSS) was performed on the mice. Polyethylene glycol (PEG) precipitation method was used to ex-tract exosomes from peripheral blood. Nanosight Tracking Analysis (NTA) was used to analyze exosomes, and quantita-tive PCR was used to detect the expression level of miR-21-5P in exosomes. Results The plasma concentration of exo-somes extracted in mTBI group was (0.84±0.24)×107 particals/mL on the 1st day after mTBI, significantly lower than(10.83±0.7)×107 particals/mL in the sham group (P<0.05); on the 28th day after mTBI, the exosome concentration in-creased compared with that on the 1st day after injury, but was still significantly lower than that in the in the sham group(P<0.05). On the 1st day after mTBI, the size of plasma exosomes in mTBI group was (131.1±2.27) nm, significantlylarger than (114.6±6.01) nm in sham group (P<0.05); on the 28th day after mTBI, the size of plasma exosomes was stillsignificantly larger than that in sham group (P<0.05). The expressoin of miR-21-5p in exosome in mTBI group on the24th day after mTBI was significantly higher than that in sham group, and the miR-21-5p level on the 28th day after mT-BI, although decreased, was still significantly higher than that in sham group (P<0.05). Conclusion After traumaticbrain injury, there are significant changes in the production and particle size of exosomes and significant increase in theexpression level of miR-21-5P. These changes persist even on the 28th day after injury. It can be seen that the produc-tion, particle size, and miR-21-5p have high value in clinical evaluation of mTBI, which are expected to become poten-tial biological indicators for evaluation of prognosis.
      【Key words】 Mild traumatic brain injury (mTBI); Plasma exosome; Production; Particle size; miR-21-5P