Let-7d在结核性脑膜炎患者脑脊液外泌体中表达的研究

发布时间：2018-03-28 10:15

本文选题：结核性脑膜炎　切入点：外泌体　出处：《郑州大学》2017年硕士论文

【摘要】：背景与目的结核性脑膜炎(Tuberculoulous meningitis,TBM)是由结核分枝杆菌引起的中枢神经系统感染性疾病,约占神经系统结核病的70%左右。其目前诊断主要依靠脑脊液常规生化、细胞学检查;然而在TBM发生的早期,脑脊液常规生化、细胞学变化多不典型,因此严重影响了TBM的早期诊治,故探讨TBM的潜在诊断标志物具有重大的临床意义。外泌体(exosomes)是一种由细胞主动分泌的大小均一,直径约30~100nm的膜性囊泡小体,具有脂质双分子层结构,广泛存在于血液、尿液、唾液、脑脊液等体液中。其内含有多种蛋白质、mRNA、miRNA等,在不同细胞间进行信息传递,发生多种生物功能。外泌体在中枢神经系统中参与正常的生理条件以及介导病理状态,在中枢神经系统的疾病的发生、发展及损伤修复中起着重要作用。微小RNA(mi RNA)是一类内源性非编码的小分子单链RNA,其主要功能是从转录后水平调节基因的表达,广泛参与多种生物的生命过程。Let-7家族是miRNA重要的组成部分之一,其包含13个成员,其中let-7d在大脑中的表达水平非常高,在多种神经系统疾病中起着重要作用,可能成为某些疾病的潜在生物学标志物。本研究通过分离临床上常见的病毒性、化脓性、结核性脑膜炎患者以及正常对照者脑脊液中的外泌体,来探讨let-7d在四种脑脊液外泌体中的表达水平,以寻求TBM新的潜在诊断标志物。材料与方法随机收集我院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)以及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,采用实时定量PCR法检测其let-7d表达水平。结果1.三种脑炎患者及正常人脑脊液中均发现外泌体,电镜下呈圆形、椭圆形或茶托状外形,直径约30~100nm,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,差异具有统计学意义(P0.05)。3.let-7d表达水平在结脑组、化脑组与对照组相比均明显下调,差异具有统计学意义(P0.05);病脑组与对照组相比也下调,但差异无统计学意义(P0.05)。4.let-7d表达水平在化脑组与结脑组相比明显下调,差异具有统计学意义(P0.05)。5.let-7d表达水平在结脑组与病脑组相比明显下调,差异具有统计学意义(P0.05)。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,通过比较三种脑炎以及正常对照者外泌体中let-7d的表达水平,发现在TBM患者中let-7d的表达水平显著下调,为其诊断提供潜在的生物学标志物。
[Abstract]:Background & objective: tuberculous meningitis (TBMM) is an infectious disease of the central nervous system caused by Mycobacterium tuberculosis, which accounts for about 70% of the tuberculosis of the nervous system. The diagnosis of TBMM mainly depends on routine biochemical and cytological examination of cerebrospinal fluid (CSF). However, in the early stage of TBM, the routine biochemical and cytological changes in cerebrospinal fluid (CSF) were atypical, which seriously affected the early diagnosis and treatment of TBM. Therefore, it is of great clinical significance to explore the potential diagnostic markers of TBM. Exocrine exosomes are a kind of membranous vesicles with the diameter of about 30~100nm, which is homogenous in size and active secreted by cells. It has a lipid bimolecular structure and widely exists in blood and urine. Saliva, cerebrospinal fluid and other body fluids contain a variety of proteins, such as mRNA-miRNA, which transmit information between different cells and produce a variety of biological functions. The exocrine body participates in normal physiological conditions and mediates the pathological state in the central nervous system. MicroRNAs play an important role in the development, development and repair of central nervous system diseases. MicroRNAs are a class of endogenous non-coding single-stranded RNAs, whose main function is to regulate the expression of genes at post-transcriptional level. The Let-7 family is one of the most important components of miRNA, which consists of 13 members, in which let-7d is highly expressed in the brain and plays an important role in a variety of neurological diseases. This study was conducted by isolating secretions from cerebrospinal fluid in patients with clinically common viral, suppurative, tuberculous meningitis and normal controls. To study the expression level of let-7d in four kinds of cerebrospinal fluid exocrine, Materials and methods Fifteen patients with tuberculous meningitis (tuberculous meningitis) were randomly collected from 15 patients with tuberculous meningitis (n = 20) with viral meningitis (n = 10) with suppurative meningitis (n = 10). Cerebrospinal fluid (CSF) of 20 normal cerebrospinal fluid patients (control group) were isolated and purified by ExoQuick Precipitation extraction method. Results 1. Exocrine bodies were found in cerebrospinal fluid of three kinds of encephalitis patients and normal subjects. The expression of let-7d in cerebrospinal fluid exocrine in tuberculous meningitis patients was significantly lower than that in control group, and the difference was statistically significant (P 0.05) .3.let-7d expression level of let-7d.2.The expression level of let-7d in tuberculous meningitis group was significantly lower than that in control group, and the expression level of let-7d in tuberculous meningitis group was significantly lower than that in control group. Compared with the control group, the expression of P0.05T in the control group was significantly lower than that in the control group (P 0.05), and the expression level of P0.05U. 4.let-7 d in the diseased brain group was lower than that in the control group, but there was no significant difference between the control group and the control group (P 0.05), and the expression level was significantly decreased in the control group (P < 0.05). The expression level of P0.05, 5.let-7d was significantly down-regulated in the nodal brain group compared with the diseased brain group, and the difference was statistically significant (P 0.05). Conclusion the exocrine can be separated from the cerebrospinal fluid of the infectious diseases of the central nervous system. By comparing the expression of let-7d in the exocrine of three encephalitis and normal controls, it was found that the expression of let-7d was significantly down-regulated in patients with TBM, which provided a potential biomarker for the diagnosis of encephalitis.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R529.3

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