【摘要】 目的 通过高通量测序技术检测颅脑损伤（TBI）患者外泌体microRNA（miRNA）的表达变化及探讨其临床意义。方法 收集2020年1月至2022年12月期间东莞市厚街医院急诊科的颅脑损伤患者15例为颅脑损伤组，选择13例同期妇科、五官科和骨科的志愿者为对照组，提取外泌体，用透射电镜鉴定外泌体的形态、纳米流式检测仪分析外泌体粒径及Western blot检测标志蛋白鉴定外泌体。每组选择3例进行miRNA测序，筛选差异表达的基因，对靶基因进行GO分析和KIGG分析。在脑脊液和血清样本中用实时荧光定量PCR方法进行验证。结果 成功从脑脊液中提取外泌体，透射电镜下观察到呈椭圆形双层膜结构的小囊泡；对照组和TBI组的平均直径分别为73.12 nm和75.38 nm，浓度分别为4.64×10⁹paritcles/ml和1.49×10¹¹paritcles/mL;Western blot检测出脑脊液中外泌体标记蛋白TGS101和CD9。测序结果显示共8个差异表达的miRNA，其中2个表达上调，6个表达下调。验证结果表明颅脑损伤患者脑脊液外泌体miR-29a-3P表达上调2.33倍，差异有统计学意义（t=3.603,P<0.05）,miR-31-5P表达上调2.05倍，差异有统计学意义（t=2.273,P<0.05）,miR-199a-5P表达下调0.32倍，差异无统计学意义（t=0.09,P>0.05）；血清外泌体中miR-29a-3P表达上调1.95倍，差异有统计学意义（t=2.11,P<0.05）,miR-31-5P表达上调2.01倍，差异有统计学意义（t=2.309,P<0.05）,miR-199a-5P表达下调0.34倍，差异有统计学意义（t=2.825,P<0.05）。结论 外泌体中miR-29a-3P和miR-31-5P有潜力作为辅助诊断TBI的生物标志物。更多还原

【Abstract】 Objective To detect the expression changes of exosomal microRNA（miRNA）in patients with traumatic brain injury（TBI）by high-throughput sequencing technology and to explore its clinical significance. Methods Fifteen patients with traumatic brain injury in the emergency department of Dongguan Houjie Hospital from January 2020 to December 2022 were selected as the traumatic injury group,and thirteen volunteers from gynecology,ophthalmology and orthopedics were selected as the control group during the same period. Exosomes were extracted to identify the morphology of exosomes by transmission electron microscopy,analyze the particle size of exosomes by nano-flow detector and identify exosomes by Western blot detection of marker proteins. Three patients were selected for miRNA sequencing in each group. Further RNA was extracted,miRNA sequencing and analysis were performed,differentially expressed genes were screened,and GO analysis and KEGG analysis were performed on their target genes. Validation was performed using real-time PCR methods in cerebrospinal fluid and serum samples. Results Exosomes were successfully extracted from the cerebrospinal fluid. Small vesicles with an oval bilayer membrane structure were observed under a transmission electron microscopy. The average diameters of the control group and the TBI group were 73.12 nm and 75.38 nm,and the concentrations were 4.64 × 10⁹paritcles/mL and 1.49 × 10¹¹paritcles/mL,respectively. Western blot detected exosome marker proteins TGS101 and CD9 in cerebrospinal fluid. The sequencing results showed a total of 8 differentially expressed miRNAs,of which 2 were up-regulated and 6 were down-regulated.The verification results showed that the expression of miR-29a-3P in cerebrospinal fluid exosomes was upregulated by 2.33 times,the difference was statistically significant（t=3.603,P<0.05）,miR-31-5P expression by 2.05 times,the difference was statistically significant（t=2.273,P<0.05）,and miR-199a-5P expression by 0.32times,and the difference was not statistically significant（t=0.099,P>0.05）. The expression of miR-29a-3P in serum was upregulated by 1.95 times,the difference was statistically significant（t=2.11,P<0.05）,miR-31-5P expression by 2.01 times,the difference was statistically significant（t=2.309,P<0.05）,and miR-199a-5P expression by 0.34 times,the difference was statistically significant（t=2.825,P<0.05）. Conclusion miR-29a-3P and miR-31-5P in exosomes have potential as biomarkers for auxiliary diagnosis of TBI.更多还原