【作者】 郭斌；

【导师】 贾延劼；

【作者基本信息】 郑州大学 ， 神经病学（专业学位）， 2018， 硕士

【摘要】 背景与目的:结核病目前仍然是一个全球性的公众健康问题,而这其中最致命和致残的结核病形式是结核性脑膜炎(tuberculous meningitis TBM),保守估计每年超过约100,000例新发病例。结核性脑膜炎是一种由结核分支杆菌通过血液传播扩散引起的非化脓性脑脊膜的炎性疾病。然而,近年来由于结核杆菌的基因突变、抗结核杆菌药物的研发迟滞、耐药性结核杆菌的出现等,相关报道显示国内外结核性脑膜炎的发病率和死亡率并未明显降低。由于结核性脑膜炎的误诊及延迟治疗十分严重,早期确诊具有重要的临床意义。病毒性脑膜炎是神经内科常见的颅内感染性疾病,由一种或多种病毒感染引起的脑膜急性或亚急性炎性疾病。主要表现为发烧、头痛、呕吐、抽搐、意识障碍等症状。随着细菌性脑膜炎的发病率的下降,病毒性脑膜炎成为临床最常见的无菌性脑膜炎。临床上柯萨奇病毒、脊髓灰质炎病毒、埃可病毒等肠道病毒感染多见,除此之外还有腺病毒、巨细胞病毒、疱疹病毒等其他病毒感染,随诊分子诊断学的临床应用,大大提高了病毒性脑膜炎的病原体的检测速度。外泌体是一种可由多种细胞分泌到胞外的纳米级囊泡体,广泛存在于患者血液、脑脊液、尿液以及胸腔积液等不同体液中,含有丰富的蛋白质、脂质、核酸等物质,作为一种新型的细胞物质交换、信息传递载体,不仅参与中枢神经系统正常的生理过程,同时也参与中枢神经系统疾病的发生发展、损伤修复等过程。微小RNA(miRNA)是细胞内大小约21到25个核苷酸构成的非编码RNA,由内源基因编码,具有高度保守性,主要与靶基因mRNA相结合,是主要的基因表达调控分子之一。Let-7家族是第一个已知的人类miRNA,通过干预细胞的分裂、增殖以及分化、凋亡等过程,进而对靶基因的表达进行调控。而let-7b作为let-7家族中的成员之一,其在神经系统中的非正常表达与神经系统多种疾病密切相关。本研究通过分离提取结核性脑膜炎、病毒性脑膜炎及正常对照者脑脊液外泌体中let-7b,来观察let-7b的表达水平,探寻脑膜炎新的生物学诊断标志物。材料与方法:收集我院神经科2017-2018年期间住院的15例结核性脑膜炎患者、15例病毒性脑膜炎患者、15例正常脑脊液患者,将其分组为结脑组、病脑组、对照组,收集5mL脑脊液标本。采取ExoQuick Precipitation提取剂分离提纯外泌体,采用透射电子显微镜观察不同组外泌体形态,应用qRT-PCR法检测各组脑脊液外泌体中let-7b相对表达水平。结果:1.结核性脑膜炎和病毒性脑膜炎及对照组脑脊液中均可以检测到外泌体,透射电镜下呈圆形或椭圆形,直径约30~100nm。荧光定量PCR检测发现3组脑脊液外泌体中均可检测到let-7b。2.结脑组脑脊液外泌体中let-7b的表达显著低于对照组,差异具有明显统计学意义(P<0.05)。3.病脑组与对照组相比,病脑组let-7b表达下调,差异具有统计学意义(P<0.05)。4.结脑组与病脑组相比,结脑组let-7b表达下调,但差异无统计学意义(P>0.05)。结论：结核性脑膜炎、病毒性脑膜炎及正常对照组中均可提纯到外泌体。let-7b在结核性脑膜炎患者脑脊液外泌体中表达下调,可作为该疾病的诊断及相关脑炎的鉴别诊断的潜在生物学标志物,但其具体的调控作用机制、结核性脑膜炎疾病治疗前后的变化仍需进一步的探索研究。更多还原

【Abstract】 BackgroundTuberculosis is still a global public health problem,and the most deadly and disabling form of tuberculosis is tuberculous meningitis(TBM).It is conservatively estimated that more than 100,000 new cases occur every year.TBM is a non-suppurative inflammatory disease of the meninges and spinal membrane caused by dissemination of Mycobacterium tuberculosis through the blood.In recent years,with the genetic mutation of Mycobacterium tuberculosis,the delay in the development of antituberculosis drugs,and the emergence of drug-resistant tuberculosis,the incidence and mortality of TBM have not been significantly reduced at home and abroad.Because the misdiagnosis and delay diagnosis of TBM is very serious,early diagnosis has an important clinical significance.Viral meningitis is a group of meningeal acute inflammatory diseases caused by various viral infections.Mainly manifested as fever,headache,vomiting and meningeal irritation.With the decline in the incidence of bacterial meningitis,viral meningitis has become the most common aseptic meningitis in the clinic.Most of them are enterovirus infections,such as Coxsackie virus,Echo virus,polio virus,and herpes simplex virus,cytomegalovirus,and adenovirus infections.Clinical applicationof follow-up molecular diagnostics has greatly increased the detection rate of viral meningitis pathogens.Exosome is a nanoscale vesicle that can be secreted into the extracellular space by a variety of cells.widely existing in the blood,urine,cerebrospinal fluid and other body fluids,containing protein,lipid and nucleic acid rich material.As a new type of cell material exchange and information transmission carrier.It not only participates in the normal physiological process of the central nervous system,but also participates in the process of development,injury repair and other process of the central nervous system diseases.MicroRNA(miRNA)is a small endogenous non-coding RNA consisting of about 21-25 nucleotides,which is highly conserved.It is mainly associated with target gene mRNA and is one of the major gene expression regulatory molecular.The Let-7 family is the first known human miRNA that participates in processes such as cell proliferation,differentiation,and apoptosis by regulating gene expression.However,let-7b is a member of the let-7 family,and its abnormal expression in the nervous system is closely related to a variety of diseases of the nervous system.In this study,we extracted let-7b in cerebrospinal fluid exosomes from TBM,viral meningitis and normal controls to observe the expression level of let-7b and explore new biomarkers for meningitis.Materials and methodsThe cerebrospinal fluid of 15 cases of tuberculous meningitis(TBM group),15 cases of viral meningitis(VM group),and 15 cases of normal cerebrospinal fluid(control group)were diagnosed and treated by our Department of Neurology in2017-2018.ExoQuick Precipitation extraction was used to separate and purify exosomes.Transmission electron microscopy was used to observe the morphology of different groups of exosomes and Western blotting to detect exosome markers.qRT-PCR was used to detect the expression of let-7b in cerebrospinal fluid exosomes.Results1.The exosomes can be detected in cerebrospinal fluid of tuberculous meningitisand viral meningitis and the control group,they are round or oval under transmission electron microscope and have a diameter of about 30 to 100 nm.Fluorescent quantitative PCR assays detected let-7b in 3 groups of cerebrospinal fluid exosomes.2.The expression level of let-7b in cerebrospinal fluid exosomes is significantly lower in patients with tuberculous meningitis than in the control group(P<0.05).3.The expression of let-7b was down-regulated in viral meningitis compared with the control group,There is statistical significance between them(P<0.05).4.The expression level of let-7 in tuberculous meningitis was lower than that in viral meningitis,but there is no statistical significance of them(P>0.05).ConclusionsExosomes can be purified from Tuberculous meningitis,viral meningitis and normal controls.The down regulation of let-7b in cerebrospinal fluid exosomes of patients with tuberculous meningitis can be used as a potential biomarker for the diagnosis of this disease and differential diagnosis of related encephalitis,but its specific regulatory mechanisms,changes before and after treatment of tuberculous meningitis disease are still needed further exploration and research.更多还原