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

Changes and significance of plasma exosome particle size and miR-21-5P expression levels after traumatic brain injury in mice

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)×10^(7)particals/mL,明显低于假手术组的(10.83±0.7)×10^(7)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。

Objective To investigate the physical characteristics of serum exosomes and the expression level of miR-21-5P in mice with mild traumatic brain injury(mTBI),and analyze the value of exosome particle size and miR-21-5P expression in the diagnosis of brain injury.Methods Twelve 8-week-old C57 mice were randomly divided into mTBI group and sham group,with 6 mice in each group.mTBI mouse models were established using Feency method.On the 1 st,7 th,and 21 st days,peripheral blood was collected from mice,and on the 3 rd,7 th,14 th,and 28 th days,neurological deficit score(mNSS)was performed on the mice.Polyethylene glycol(PEG)precipitation method was used to extract exosomes from peripheral blood.Nanosight Tracking Analysis(NTA)was used to analyze exosomes,and quantitative PCR was used to detect the expression level of miR-21-5P in exosomes.Results The plasma concentration of exosomes extracted in mTBI group was(0.84±0.24)×10^(7)particals/mL on the 1 st day after mTBI,significantly lower than(10.83±0.7)×10^(7)particals/mL in the sham group(P<0.05);on the 28 th day after mTBI,the exosome concentration increased compared with that on the 1 st day after injury,but was still significantly lower than that in the in the sham group(P<0.05).On the 1 st day after mTBI,the size of plasma exosomes in mTBI group was(131.1±2.27)nm,significantly larger than(114.6±6.01)nm in sham group(P<0.05);on the 28 th day after mTBI,the size of plasma exosomes was still significantly larger than that in sham group(P<0.05).The expressoin of miR-21-5p in exosome in mTBI group on the 24 th day after mTBI was significantly higher than that in sham group,and the miR-21-5p level on the 28 th day after mTBI,although decreased,was still significantly higher than that in sham group(P<0.05).Conclusion After traumatic brain injury,there are significant changes in the production and particle size of exosomes and significant increase in the expression level of miR-21-5P.These changes persist even on the 28th day after injury.It can be seen that the production,particle size,and miR-21-5p have high value in clinical evaluation of mTBI,which are expected to become potential biological indicators for evaluation of prognosis.