HIV患者脑脊液蛋白质组学的研究

发布时间：2018-02-25 19:05

本文关键词： HIV 脑脊液蛋白质组学二维液相色谱差异表达蛋白　出处：《华东师范大学》2013年硕士论文　论文类型：学位论文

【摘要】：自从1981年人类首次报道了人类获得性免疫缺陷综合症以来,该病已经在全球广泛盛行,超过20万的人死于艾滋。HIV-1病毒不仅损坏免疫系统,而且具有嗜神经性。从而HIV-1便成为全世界继阿尔茨海默病和血管性痴呆后的又一主要的痴呆原因。HIV相关认知障碍是一类由HIV-1感染引起的神经系统疾病,是HIV-1侵犯中枢神经系统造成病人意识、行为和运动能力改变为主要表现的综合征。严重时可危及生命。HIV相关认知障碍的具体发病机制尚不清楚。目前认为,HIV或呈游离状态或借助感染的免疫细胞跨过血脑屏障或血脑脊液屏障进入中枢神经系统,其发病主要是由HIV病毒蛋白的毒性效应、诱导毒性细胞因子的产生和自身免疫而间接引起的。而目前有关病毒的分子生物学指标如CSF中HIV的RNA水平及相关的免疫学指标在HIV相关认知障碍的诊断和疗效评估方面并无显著的应用价值。HIV相关认知障碍在临床实践中显得较为棘手,因此迫切需要筛选具有临床应用价值的新型生物标志物。脑脊液(cerebrospinal fluid, CSF)是存在于脑室及蛛网膜下腔内的一种无色透明的液体,循环流动于脑和脊髓表面,大部分由脑室脉络丛分泌。中枢神经系统病变一般会导致CSF蛋白种类的改变、某些蛋白的修饰或浓度的波动。CSF与中枢神经系统直接联系,反映了在不同病理条件下中枢神经系统的生化状态,是研究中枢神经系统疾病的一个独特的窗口。目前关于艾滋痴呆方面的蛋白质研究进展比较缓慢,而随着蛋白质组学的飞速发展,己可以采用相关技术直接筛选具有临床价值的的蛋白标志物。因此我们利用二维液相色谱(2D-LC)偶联基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)技术进行HIV相关认知障碍患者脑脊液差异蛋白质组学的研究,以筛选具有临床应用价值的新型生物标志物,对HIV相关认知障碍的早期诊断、疗效观察和预后判断等均具有明显的现实意义。 1、脑脊液蛋白的二维液相色谱分离方法建立一方面对二维液相色谱分离系统PF2D方法的稳定性、可靠性进行验证。PF2D分离系统具有良好的重复性和准确性,并且峰面积与蛋白浓度成正比,保证了后续分析脑脊液蛋白的可靠性。另一方面提取脑脊液的蛋白质进行初步的纯化定量,进行研究。采用乙醇沉淀的方法提取脑脊液的蛋白能得到浓度为0.5-2.6mg/mL的蛋白,满足2-DL分析1-5mg的蛋白上样量。 2、感染HIV的脑脊液蛋白表达差异分析根据前面建立的二维液相分离方法,我们分别对感染了HIV的脑脊液和没有感染的四种脑脊液样品进行PF-2D的二维分离,第一维的分离中检测器的波长设为280nm,每变化0.3个单位的pH进行收集,在pH从8.5下降到4.0的过程中收集到了16个一维组分。将每个一维组分分别进行二维分离,并用DeltVue软件分析两样品中相同pH变化的二维紫外吸引图,结果显示有95个峰有显著的差异,相对于对照组,HIV组有41个峰上调,有54个峰下调。再选取二维分离样品中紫外吸收值小于0.1的峰作快速银染检测,结果表明检测到了蛋白。将这些有显著差异的色谱组分委托布鲁克公司(北京)做MALDI-TOF/TOF质谱鉴定。由于该公司仪器出现问题,尚未给出最终结果。但从一个已鉴定出的蛋白,即白蛋白的结果看,本研究所分离得到的蛋白样品符合质谱鉴定的要求。
[Abstract]:HIV - 1 has become a major cause of dementia following Alzheimer ' s disease and vascular dementia . HIV - 1 has become a major cause of dementia following Alzheimer ' s disease and vascular dementia . HIV - 1 has become a major cause of dementia following Alzheimer ' s disease and vascular dementia . CSF is a colorless and transparent liquid present in the ventricular and subarachnoid space , circulating on the surface of the brain and the spinal cord , most of which is secreted by the brain plexus . The central nervous system lesions generally result in changes in the type of CSF protein , the modification of certain proteins or the fluctuation of the concentration . The CSF is directly associated with the central nervous system , which reflects the biochemical status of the central nervous system under different pathological conditions , and is a unique window for the study of the central nervous system disease . Recently , with the rapid development of Proteomics , the research of differential proteomic analysis of cerebrospinal fluid in patients with HIV - related cognitive disorders was studied by using 2D - LC coupled matrix - assisted laser desorption time - of - flight mass spectrometry ( MALDI - TOF - MS ) . 1 . Two - dimensional liquid chromatography separation method for cerebrospinal fluid protein On the one hand , the stability and the reliability of the PF2D method of the two - dimensional liquid chromatography separation system are verified . The PF2D separation system has good repeatability and accuracy , and the peak area is directly proportional to the protein concentration , so that the subsequent analysis of the reliability of the cerebrospinal fluid protein is ensured . On the other hand , the protein of the cerebrospinal fluid is extracted to obtain the protein with the concentration of 0.5 - 2.6 mg / mL , and the amount of the protein on the 2 - DL analysis of 1 - 5 mg is met . 2 . The difference of the expression of cerebrospinal fluid protein in HIV infected with HIV was analyzed according to the two - dimensional liquid - phase separation method established in the preceding paragraph . The two - dimensional separation was carried out for four kinds of cerebrospinal fluid samples with HIV infection and four kinds of cerebrospinal fluid without infection . The results showed that there were significant differences in the pH value of the first dimension . The results showed that the protein was detected by MALDI - TOF / TOF mass spectrometry .

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91;Q75

【共引文献】