本文關鍵詞： 博爾納病毒 金剛烷胺 星形膠質細胞 代謝組學　出處：《重慶醫(yī)科大學》2014年碩士論文　論文類型：學位論文

【摘要】：背景 博爾納病病毒（Borna disease virus，BDV）19世紀末在德國南部博爾納鎮(zhèn)被首次發(fā)現(xiàn)，之后20年流行病學研究證明，BDV的自然宿主極為廣泛，可以感染包括人類在內的大多數(shù)恒溫動物，人們才意識到BDV有著更為廣闊的感染區(qū)域。BDV是一種有包膜非節(jié)段性的單股負鏈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細胞中復制和轉錄的RNA病毒，因此做為典型代表被歸入單股負鏈病毒目中的Borna病毒科。該病毒具有嚴格嗜神經(jīng)細胞性，可持續(xù)慢性感染神經(jīng)細胞并且長期寄居在神經(jīng)系統(tǒng)內不被機體免疫系統(tǒng)清除，近年來，BDV的抗原、特異性抗體及RNA已頻繁的在神經(jīng)精神疾病患者的外周血單核細胞和腦組織中被檢測到，而且BDV感染羊和馬等恒溫動物后可引起冷漠、行為失常、運動失調等癥狀，這些臨床癥狀也與精神分裂以及情感障礙等疾病的臨床癥狀相似，這些研究均顯示該病毒感染與人類精神疾病緊密相關，LivBode等研究發(fā)現(xiàn)，金剛烷胺具有抗BDV病毒作用，雙向情感障礙患者服用該藥后癥狀有所緩解，但是該藥抑制BDV病毒感染機制仍未進入深入研究。 目的 應用1HNMR代謝組學方法，獲得正常對照組、BDV感染組及金剛烷胺干預組的C6細胞所有差異代謝物，比較三組之間代謝物的變化，從差異代謝產(chǎn)物水平探討B(tài)DV病理生理致病因素及金剛烷胺抗BDV病毒的作用機制。 方法 提取BDV長期感染OL細胞中的博爾納病毒，用病毒液感染C6細胞，分為正常對照組，BDV感染組以及金剛烷胺干預組，，每組三個重復。甲醇氯仿萃取法提取各組細胞內代謝物，采用核磁共振（1Hnuclear magnetic resonance,1H NMR）技術對各組細胞內代謝物進行分析，區(qū)分各組細胞內差異代謝物采用應用Q-檢驗和T-檢測，最后采用偏最小二乘判別分析（PLS-DA）對三組細胞代謝物進行模式分析。 結果 病毒滴度法測定所提BDV病毒具有一定感染能力，并且RT-PCR技術及WB實驗均從基因及蛋白水平驗證BDV感染細胞后成功進行轉錄翻譯及蛋白表達。 對正常對照組與BDV感染組提取的細胞內代謝物進行1H NMR分析，結果顯示，定量分析27種代謝產(chǎn)物，其中BDV感染引起代謝物水平升高的有20種：異亮氨酸、纈氨酸、乳酸鹽、丙氨酸、醋酸鹽、-萘乙酸、丙酮酸、谷氨酸、琥珀酸鹽、谷氨酰胺、肌酸、膽堿、肌醇、甘氨酸、酪氨酸、β-葡萄糖、苯基丙氨酸、甲酸鹽、AMP、NAD+；降低的有7個：亮氨酸、2-羥基異丁酸、天冬氨酸、焦谷氨酸、乳糖、組氨酸、ADP。差異代謝物21種，包括：異亮氨酸、纈氨酸、乳酸鹽、丙氨酸、丙酮酸、谷氨酸、琥珀酸鹽、谷氨酰胺、肌酸、肌醇、甘氨酸、酪氨酸、β-葡萄糖、苯基丙氨酸、甲酸鹽、AMP、亮氨酸、2-羥基異丁酸、天冬氨酸、乳糖、組氨酸。 用藥干預組與正常對照組無差異，但是與病毒感染組有差異的代謝物有16個，分別是：異亮氨酸、纈氨酸、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、組氨酸。 結論 結果顯示， BDV感染C6細胞后增強三羧酸循環(huán)通路活性從而控制細胞的能量代謝通路，金剛烷胺用藥后相關能量代謝物質丙酮酸、乳酸、琥珀酸降低，從而阻斷病毒的復制增殖，這些代謝產(chǎn)物的變化提示BDV影響了宿主細胞的正常代謝活動，金剛烷胺通過反轉該類代謝物水平從而達到抗病毒作用，為闡明金剛烷胺抗BDV的生化機制提供了一些線索，同時為金剛烷胺治療精神疾病研究提供相關依據(jù)。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96

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