金刚烷胺干预博尔纳病毒感染C6细胞的代谢组学研究

发布时间：2018-02-26 05:35

本文关键词： 博尔纳病毒金刚烷胺星形胶质细胞代谢组学　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景博尔纳病病毒（Borna disease virus，BDV）19世纪末在德国南部博尔纳镇被首次发现，之后20年流行病学研究证明，BDV的自然宿主极为广泛，可以感染包括人类在内的大多数恒温动物，人们才意识到BDV有着更为广阔的感染区域。BDV是一种有包膜非节段性的单股负链RNA病毒（Non-segmented negative-strand RNA virus,NNR病毒），由于它是目前唯一能在被感染细胞中复制和转录的RNA病毒，因此做为典型代表被归入单股负链病毒目中的Borna病毒科。该病毒具有严格嗜神经细胞性，可持续慢性感染神经细胞并且长期寄居在神经系统内不被机体免疫系统清除，近年来，BDV的抗原、特异性抗体及RNA已频繁的在神经精神疾病患者的外周血单核细胞和脑组织中被检测到，而且BDV感染羊和马等恒温动物后可引起冷漠、行为失常、运动失调等症状，这些临床症状也与精神分裂以及情感障碍等疾病的临床症状相似，这些研究均显示该病毒感染与人类精神疾病紧密相关，LivBode等研究发现，金刚烷胺具有抗BDV病毒作用，双向情感障碍患者服用该药后症状有所缓解，但是该药抑制BDV病毒感染机制仍未进入深入研究。目的应用1HNMR代谢组学方法，获得正常对照组、BDV感染组及金刚烷胺干预组的C6细胞所有差异代谢物，比较三组之间代谢物的变化，从差异代谢产物水平探讨BDV病理生理致病因素及金刚烷胺抗BDV病毒的作用机制。方法提取BDV长期感染OL细胞中的博尔纳病毒，用病毒液感染C6细胞，分为正常对照组，BDV感染组以及金刚烷胺干预组，，每组三个重复。甲醇氯仿萃取法提取各组细胞内代谢物，采用核磁共振（1Hnuclear magnetic resonance,1H NMR）技术对各组细胞内代谢物进行分析，区分各组细胞内差异代谢物采用应用Q-检验和T-检测，最后采用偏最小二乘判别分析（PLS-DA）对三组细胞代谢物进行模式分析。结果病毒滴度法测定所提BDV病毒具有一定感染能力，并且RT-PCR技术及WB实验均从基因及蛋白水平验证BDV感染细胞后成功进行转录翻译及蛋白表达。对正常对照组与BDV感染组提取的细胞内代谢物进行1H NMR分析，结果显示，定量分析27种代谢产物，其中BDV感染引起代谢物水平升高的有20种：异亮氨酸、缬氨酸、乳酸盐、丙氨酸、醋酸盐、-萘乙酸、丙酮酸、谷氨酸、琥珀酸盐、谷氨酰胺、肌酸、胆碱、肌醇、甘氨酸、酪氨酸、β-葡萄糖、苯基丙氨酸、甲酸盐、AMP、NAD+；降低的有7个：亮氨酸、2-羟基异丁酸、天冬氨酸、焦谷氨酸、乳糖、组氨酸、ADP。差异代谢物21种，包括：异亮氨酸、缬氨酸、乳酸盐、丙氨酸、丙酮酸、谷氨酸、琥珀酸盐、谷氨酰胺、肌酸、肌醇、甘氨酸、酪氨酸、β-葡萄糖、苯基丙氨酸、甲酸盐、AMP、亮氨酸、2-羟基异丁酸、天冬氨酸、乳糖、组氨酸。用药干预组与正常对照组无差异，但是与病毒感染组有差异的代谢物有16个，分别是：异亮氨酸、缬氨酸、乳酸、丙氨酸、丙酮酸、谷氨酸、琥珀酸、谷氨酰胺、肌酸、甘氨酸、葡萄糖、酪氨酸、苯丙氨酸、甲酸、乳糖、组氨酸。结论结果显示， BDV感染C6细胞后增强三羧酸循环通路活性从而控制细胞的能量代谢通路，金刚烷胺用药后相关能量代谢物质丙酮酸、乳酸、琥珀酸降低，从而阻断病毒的复制增殖，这些代谢产物的变化提示BDV影响了宿主细胞的正常代谢活动，金刚烷胺通过反转该类代谢物水平从而达到抗病毒作用，为阐明金刚烷胺抗BDV的生化机制提供了一些线索，同时为金刚烷胺治疗精神疾病研究提供相关依据。
[Abstract]:background
Borna disease virus (Borna disease, virus, BDV) at the end of nineteenth Century in the southern German town of Borna was first discovered, after 20 years of epidemiological studies have shown that the natural host of BDV is very wide and the most warm blooded animal can infect humans included, people realized that BDV has a more wide area.BDV infection is a non enveloped single strand RNA virus segmental (Non-segmented negative-strand RNA virus, NNR virus), because it is the only way to be in the infection and replication and transcription of the RNA virus, Borna virus, so do as the typical representative is classified as single stranded virus. The virus is strict. Perineural cells, nerve cells and long-term chronic infection of sustainable living in the nervous system is not clear, the immune system in recent years, BDV antigen, specific antibody and RNA have been frequent in neuropsychiatric diseases. In patients with peripheral blood mononuclear cells and brain tissue were detected, and BDV infection in sheep and horses and other warm blooded animal can cause apathy, behavioral disorders, movement disorders and other symptoms, the clinical symptoms of these diseases and clinical symptoms of schizophrenia and affective disorders and other similar, these studies showed that the virus infection closely related to human mental illness, LivBode found that amantadine has the effect of anti BDV virus, after taking the drug in patients with ease the symptoms of bipolar disorder, but the inhibition of BDV virus infection mechanism has not yet entered the study.
objective
Application of 1HNMR metabonomics method, obtained the normal control group, BDV infection group and amantadine intervention group C6 cells of all the differences between the three groups comparison of metabolites, metabolite changes, to explore the mechanisms of BDV pathogenesis and pathophysiology of amantadine against BDV virus from differences in metabolite levels.
Method
The extraction of long-term BDV infection in OL cells with Borna disease virus, retrovirus infected C6 cells were divided into normal control group, BDV infection group and amantadine intervention group, each with three replicates. The intracellular metabolite extraction of methanol chloroform extraction method, using nuclear magnetic resonance (1Hnuclear magnetic resonance, 1H NMR) on cell metabolites in the analysis, distinguish the difference of intracellular metabolites were used Q- test and T- test, finally using partial least squares discriminant analysis (PLS-DA) pattern analysis is carried out on three groups of cellular metabolites.
Result
Virus titer method has certain infection ability to detect the BDV virus, and RT-PCR technology and WB test have successfully transcribed and expressed protein from BDV infected cells from gene and protein level.
1H NMR analysis of metabolites in cell group and BDV infection group from normal control in analysis results showed that 27 metabolites including quantitative infection caused by elevated levels of BDV metabolites are of 20 kinds: isoleucine, valine, lactate, alanine, naphthalene acetic acid, acetate, pyruvate, succinate, glutamine, glutamate. Creatine, choline, inositol, tyrosine, glycine, beta glucose, phenylalanine, formate, AMP, NAD+; reduced 7: leucine, 2- hydroxy isobutyric acid, aspartic acid, pyroglutamic acid, milk sugar, histidine, ADP. different metabolites 21 kinds, including: isoleucine, valine, lactate, alanine, pyruvate, glutamate, succinate, glutamine, creatine, myo inositol, tyrosine, glycine, beta glucose, phenylalanine, formate, AMP, leucine, 2- hydroxy isobutyric acid, aspartic acid, lactose, histidine.
No differences in drug intervention group and normal control group, but there are differences in the virus infection group and 16 metabolites, namely: isoleucine, valine, lactate, pyruvate, alanine, glutamic acid, succinic acid, glutamine, creatine, glycine, phenylalanine, tyrosine, glucose, lactose, formic acid, histidine.
conclusion
The results showed that BDV infected C6 cells enhanced energy metabolism pathways of three tricarboxylic acid cycle pathway to control cell, amantadine drug related energy metabolites pyruvic acid, lactic acid, succinic acid decreased, thereby blocking viral replication, variation of these metabolites suggests that BDV affects the normal metabolism of host cells, adamantane through this kind of reversal amine metabolite levels so as to achieve the antiviral effect, provide some clues for elucidating the biochemical mechanisms of amantadine resistance BDV, and provide the basis for the study on the treatment of mental illness of amantadine.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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