结核性脑膜炎患者脑脊液外泌体中let-7d表达的研究

摘要

目的 探讨结核性脑膜炎(tuberculoulous meningitis,TBM)患者脑脊液外泌体的分离及外泌体中let-7d的表达水平及其意义.方法 收集我院15例结核性脑膜炎患者(结脑组,TBM组),20例病毒性脑膜炎患者(病脑组,VM组),10例化脓性脑膜炎患者(化脑组,PM组)及20例正常脑脊液患者(对照组)的脑脊液,采取ExoQuick Precipitation提取法分离并纯化外泌体,通过透射电镜观察其形态,采用实时荧光定量PCR法检测其let-7 d表达水平.结果 (1)脑脊液中的外泌体,电镜下呈圆形或茶托状外形,直径40~100 nm,实时定量PCR法检测发现其内含有不同程度表达水平的let-7d;(2)结核性脑膜炎患者脑脊液外泌体中let-7d的表达水平显著低于对照组,差异有统计学意义(P<0.05);(3)let-7d表达水平结脑组、化脑组与对照组相比均明显下调,差异有统计学意义(P<0.05);病脑组与对照组相比也下调,但差异无统计学意义(P>0.05);(4)let-7d表达水平在化脑组与结脑组相比明显下调,差异有统计学意义(P<0.05);(5)let-7d表达水平在结脑组与病脑组相比明显下调,差异有统计学意义(P<0.05).结论 中枢神经系统感染性疾病的脑脊液中可分离出外泌体,通过比较3种脑炎外泌体中let-7d的表达水平,发现在TBM患者中let-7 d的表达水平显著下调,为其诊断提供潜在的生物学标志物.%Objective To investigate the exosomes isolation from cerebrospinal fluid(CSF)in patients with tuberculous meningitis(TBM)and the expression of let-7d in exosomes.Methods The cerebrospinal fluid from 15 cases with tuberculous men-ingitis(TBM group),20 cases with viral meningitis(VM group),10 cases with purulent meningitis patients(PM group)and 20 normal cases(control group)was collected.ExoQuick Precipitation was used to isolate and purify exosomes and transmission elec-tron microscopy was applied to observe the morphology.Eventually,the expression of let-7d was detected by real-time fluorescence quantitative PCR.Results (1)The exosomes in cerebrospinal fluid were round or cup-shaped,and the diameter was about 40~100nm.Real-time quantitative PCR found varying expression levels of let-7d in exosomes.(2)The expression level of let-7d in TBM group was significantly lower than that in control group(P<0.05).(3)The TBM and PM groups showed higher down-regu-lation of the expression level of let-7d compared with the control group(P<0.05).(4)The expression level of let-7d in the PM group was significantly down-regulated relative to the TBM group(P<0.05).(5)Significant down-regulation of expression level of let-7d was found in TBM group compared with VM group(P<0.05).Conclusion Exosomes can be isolated from cerebrospinal fluid in infectious diseases of the central nervous system.By comparing the expression level of let-7d in three sorts of encephslitis, we find that the expression of let-7d in TBM patients is significantly down-regulated,providing a potential biomarker for diagno-sis.