臺灣博碩士論文加值系統

日本腦炎病毒是引起日本腦炎的致病因，分類上屬於黃病毒科（Flaviviridae）黃病毒屬（Flavivirus）。日本腦炎病毒藉由蚊子叮咬進入寄主體內，臨床病例發現患者主要有神經症狀而感染侵犯部位則是中樞神經系統。感染部位的組織病理變化方面也發現，感染區的神經細胞有功能失常及死亡現象，並伴隨有顯著的發炎反應，免疫細胞浸潤。以往研究發現，有些病毒會引起一些神經退化性疾病，比如Human Immunodeficiency Virus, HIV所引起的痴呆(Dementia)，HIV不會感染神經元細胞，但會感染活化神經膠細胞，促使神經膠細胞釋放神經傷害物質，造成神經元細胞死亡。此一間接傷害模式在黃病毒屬研究資料尚少。近來有文獻顯示，日本腦炎病毒感染活體動物的組織切片，可以觀察到神經膠細胞有活化的現象。因此我們的研究重點擺在日本腦炎病毒感染活化神經膠細胞的探討。在我們的研究發現，日本腦炎病毒確實會感染神經膠細胞，而且會促使被感染的神經膠細胞型態改變，也會促使細胞產生一氧化氮以及細胞激素/趨化激素(IL-1β, IL-6, TNF-α, RANTES)，並且會促進微神經膠細胞增生。臨床上日本腦炎患者會出現痙攣、肌強直、異常行為等症狀近似興奮性神經毒性。神經傳導因子麩胺酸(Glutamate)的釋放會誘發鈣離子移動，造成興奮性神經毒性。在我們的研究，日本腦炎病毒確實會促使微神經膠細胞增加麩胺酸的釋放。藉由麩胺酸轉運體(Glutamate transpoter)活性抑制劑(PDC，DL-TBOA)的應用來分析堆積的原因，發現日本腦炎病毒所引起的麩胺酸釋放，並非是透過麩胺酸轉運體這一類通道。另外我們的研究也發現，麩胺酸在細胞內的產量也會因日本腦炎病毒感染而增加。而細胞內麩胺酸量增加的原因，我們證實主因是日本腦炎病毒促進麩醯胺酸酶(Glutaminase)的表現，致使細胞內麩胺酸產量增加。參考文獻，以脂多醣體(LPS)刺激微神經細胞造成麩胺酸大量釋放的模式中，其最主要的原因是TNF-α引起的。我們的研究證明，日本腦炎病毒所造成的麩胺酸釋放，TNF-α確實有參與部分調控，但並非全由TNF-α造成。我們證實另外還有一氧化氮，但不是PGE2有參與調控日本腦炎病毒感染微神經膠細胞造成的麩胺酸釋放。我們更進一步發現訊號傳遞分子PKC也有參與其中。以上，日本腦炎病毒感染活化現象所產生的發炎因子以及麩胺酸的釋放皆很有可能會間接造成神經傷害。

Japanese encephalitis virus (JEV), a member of the family Flaviviridae, which causes acute encephalitis in human. JEV targets the CNS, clinically manifesting with fever, headache, vomiting, signs of meningeal irritation, and altered consciousness, leading to high mortality and neurological sequelae in some of those who survive. Though neurological disorder caused by conventional viruses are often characterized by evidence of immune system recognition and the presence of inflammatory components among the neuropathological changes, the mechanisms by which these viruses cause neurological disease are not fully understood. In many cases, the virus is probably not directly involved in the destruction of brain tissue but many cause damage indirectly by triggering cell mediated immune response by activating glial cells. In our studies, morphological change and pro-inflammatory cytokine production were found in JEV-infected glial cells. In addition, JEV-infected microglia significantly elevated extracellular level of glutamate, a major excitotoxic neurotransmitter in the CNS. We have demonstrated that TNF-α, NO and PKC might involve in regulating glutamate release. JEV-infected microglia elevated intracellular glutamate level through the up-regulating glutaminase. Generally, cytokine production and glutamate release are well recognized events in glial activation. Our findings suggest that JEV infection induces glial activation. In conclusion, the relationship of JEV infection, neuroglia activation, and inflammatory responses are highly relative. The inflammatory responses of activated neuroglia might play a role in JEV-induced neurotoxicity.

目錄 i
縮寫表 v
中文摘要 vi
英文摘要 viii
一、緒論 1
1-1、前言 1
1-2、日本腦炎流行病學 1
1-3、日本腦炎臨床症狀和治療 3
1-4、日本腦炎病毒分子特徵 5
1-5、中樞神經系統細胞組成 8
1-6、病毒與中樞神經系統 11
1-7、麩胺酸與中樞神經系統 12
1-8、研究背景與目的 13
二、實驗材料及方法 15
2-1、實驗材料 15
2-2、病毒株的製備:日本腦炎病毒增殖 16
2-3、病毒定量:病毒溶斑試驗(Plaque assay) 16
2-4、初代神經細胞培養(Primary cell culture) 17
2-5、西方墨點法(Western blot) 20
2-6、RT-PCR 22
2-7、測量一氧化氮的生成 24
2-8、細胞免疫染色 24
2-9、酵素連結免疫分析(ELISA) 25
2-10、乳酸脫氫酶釋出試驗(LDH efflux assay) 26
2-11、粒線體活性分析(MTS assay) 26
2-12、細胞增生分析 27
2-13、高效液相層析技術(HPLC) 27
2-14、統計分析 29
三、實驗結果 30
3-1、日本腦炎病毒感染對神經元細胞傷害的分析 30
3-2、日本腦炎病毒對混合神經膠細胞感染特性分析 30
3-3、日本腦炎病毒感染混合神經膠細胞後的細胞型態分
析 31
3-4、日本腦炎病毒感染混合神經膠細胞後引發一氧化氮
的產生 31
3-5、日本腦炎病毒感染混合神經膠細胞後的細胞激素產
生情形 32
3-6、日本腦炎病毒感染混合神經膠細胞後，PGE2與
COX-2產生情形 33
3-7、日本腦炎病毒感染不同組成初代神經細胞後的麩胺
酸釋放情形 34
3-8、感染日本腦炎病毒促使微神經膠細胞增生 34
3-9、日本腦炎病毒感染微神經膠細胞後麩胺酸在不同時
間釋放情形 35
3-10、麩胺酸運輸體對麩胺酸釋放影響之分析 35
3-11、麩醯胺酸酶對麩胺酸釋放之影響分析 36
3-12、脂多醣體刺激微神經膠細胞對麩胺酸釋放之影響
分析 37
3-13、TNF-α對麩胺酸釋放之影響分析 37
3-14、一氧化氮對麩胺酸釋放之影響分析 38
3-15、COX-2對麩胺酸釋放之影響分析 39
3-16、PKC對麩胺酸釋放之影響分析 39
四、討論 41
五、結論 51
六、圖表 52
圖3-1、日本腦炎病毒感染神經元/神經膠細胞後對神經元 細胞傷害的分析 52
圖3-2、日本腦炎病毒感染神經元/神經膠細胞後乳酸脫氫酶釋出分析 53
圖3-3、混合神經膠細胞經日本腦炎病毒感染後，分析細胞乳酸脫氫酶釋出情形，以及分析病毒E片段 mRNA及NS1蛋白質的表現 54
圖3-4、日本腦炎病毒感染混合神經膠細胞後，觀察GFAP陽性細胞形態 55
圖3-5、日本腦炎病毒感染混合神經膠細胞分析後，ED-1陽性細胞形態轉變 56
圖3-6、日本腦炎病毒感染混合神經膠細胞後，一氧化氮與誘發性一氧化氮合成酶的產生情形 57
圖3-7、日本腦炎病毒感染混合神經膠細胞後的細胞激素mRNA及蛋白產生情形 58
圖3-8、日本腦炎病毒感染混合神經膠細胞後，PGE2產生情形。以及日本腦炎病毒感染微神經膠細胞，COX-2的mRNA與蛋白產生情形 59
圖3-9、日本腦炎病毒感染混合神經膠細胞、神經元/神經
膠細胞、微神經膠細胞、星狀神經膠細胞後各種
細胞的麩胺酸釋放情形 60
圖3-10、感染日本腦炎病毒促使微神經膠細胞增生 61
圖3-11、日本腦炎病毒感染微神經膠細胞後麩胺酸在不
同時間釋放情形 62
圖3-12、麩胺酸運輸體抑制劑對麩胺酸釋放之影響 63
圖3-13、日本腦炎病毒感染對細胞內麩胺酸產生之影響與麩醯胺酸對麩胺酸釋放之影響 64
圖3-14、日本腦炎病毒感染微神經膠細胞後麩醯胺酸酶mRNA表現情形與麩醯胺酸酶抑制劑對麩胺酸釋放之影響 65
圖3-15、細菌內毒素脂多醣體刺激微神經膠細胞後麩胺酸釋放情形 66
圖3-16、TNF-α抗體作為中和抗體對麩胺酸釋放之影響 67
圖3-17、一氧化氮合成酶抑制劑對麩胺酸釋放之影響 68
圖3-18、日本腦炎病毒感染誘發COX-2基因表現以及COX-2抑制劑對麩胺酸釋放之影響 69
圖3-19、PKC抑制劑對麩胺酸釋放之影響 70
七、參考文獻 71

Adamson, D. C., B. Wildemann, et al. (1996). "Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41." Science 274(5294): 1917-21.
Ankarcrona, M., J. M. Dypbukt, et al. (1995). "Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function." Neuron 15(4): 961-73.
Bianchi, R., C. Adami, et al. (2007). "S100B binding to RAGE in microglia stimulates COX-2 expression." J Leukoc Biol 81(1): 108-18.
Boven, L. A., L. Montagne, et al. (2000). "Macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and RANTES mRNA semiquantification and protein expression in active demyelinating multiple sclerosis (MS) lesions." Clin Exp Immunol 122(2): 257-63.
Brannan, C. A. and M. R. Roberts (2004). "Resident microglia from adult mice are refractory to nitric oxide-inducing stimuli due to impaired NOS2 gene expression." Glia 48(2): 120-31.
Brown, G. C. and A. Bal-Price (2003). "Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria." Mol Neurobiol 27(3): 325-55.
Chambers, T. J., C. S. Hahn, et al. (1990). "Flavivirus genome organization, expression, and replication." Annu Rev Microbiol 44: 649-88.
Chang, Y. S., C. L. Liao, et al. (1999). "Membrane permeabilization by small hydrophobic nonstructural proteins of Japanese encephalitis virus." J Virol 73(8): 6257-64.
Chen, C. J., J. H. Chen, et al. (2004). "Upregulation of RANTES gene expression in neuroglia by Japanese encephalitis virus infection." J Virol 78(22): 12107-19.
Choi, D. W. (1988). "Glutamate neurotoxicity and diseases of the nervous system." Neuron 1(8): 623-34.
Choi, D. W. (1992). "Excitotoxic cell death." J Neurobiol 23(9): 1261-76.
Croen, K. D. (1993). "Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication." J Clin Invest 91(6): 2446-52.
Curthoys, N. P. and M. Watford (1995). "Regulation of glutaminase activity and glutamine metabolism." Annu Rev Nutr 15: 133-59.
Dietrich, J., B. M. Blumberg, et al. (2004). "Infection with an endemic human herpesvirus disrupts critical glial precursor cell properties." J Neurosci 24(20): 4875-83.
Drent, M., N. A. Cobben, et al. (1996). "Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation." Eur Respir J 9(8): 1736-42.
Eglitis, M. A. and E. Mezey (1997). "Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice." Proc Natl Acad Sci U S A 94(8): 4080-5.
Fonseca, L. L., M. A. Monteiro, et al. (2005). "Cultures of rat astrocytes challenged with a steady supply of glutamate: new model to study flux distribution in the glutamate-glutamine cycle." Glia 51(4): 286-96.
German, A. C., K. S. Myint, et al. (2006). "A preliminary neuropathological study of Japanese encephalitis in humans and a mouse model." Trans R Soc Trop Med Hyg 100(12): 1135-45.
Giulian, D. and T. J. Baker (1986). "Characterization of ameboid microglia isolated from developing mammalian brain." J Neurosci 6(8): 2163-78.
Habu A, M. Y., Ogasa A, Fujisaki Y. (1977). "Disorder of spermatogenesis and viral discharge into semen in boars infected with Japanese encephalitis virus." Uirusu. Journal of virology 27(1): 21-6.
Hanisch, U. K. (2002). "Microglia as a source and target of cytokines." Glia 40(2): 140-55.
Hase, T., P. L. Summers, et al. (1987). "Maturation process of Japanese encephalitis virus in cultured mosquito cells in vitro and mouse brain cells in vivo." Arch Virol 96(3-4): 135-51.
Hull, M., B. Muksch, et al. (2006). "Amyloid beta peptide (25-35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes." Neurochem Int 48(8): 663-72.
Jimenez, J. L., J. Gonzalez-Nicolas, et al. (2001). "Regulation of human immunodeficiency virus type 1 replication in human T lymphocytes by nitric oxide." J Virol 75(10): 4655-63.
Kan, H., Z. Xie, et al. (2000). "HIV gp120 enhances NO production by cardiac myocytes through p38 MAP kinase-mediated NF-kappaB activation." Am J Physiol Heart Circ Physiol 279(6): H3138-43.
Ke, Z. J. and G. E. Gibson (2004). "Selective response of various brain cell types during neurodegeneration induced by mild impairment of oxidative metabolism." Neurochem Int 45(2-3): 361-9.
Kim, W. G., R. P. Mohney, et al. (2000). "Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia." J Neurosci 20(16): 6309-16.
Kimura, T., J. Kimura-Kuroda, et al. (1994). "Analysis of virus-cell binding characteristics on the determination of Japanese encephalitis virus susceptibility." Arch Virol 139(3-4): 239-51.
Kleinert, H., T. Wallerath, et al. (1998). "Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-kappaB-signaling pathways." Br J Pharmacol 125(1): 193-201.
Koci, M. D., L. A. Kelley, et al. (2004). "Astrovirus-induced synthesis of nitric oxide contributes to virus control during infection." J Virol 78(3): 1564-74.
Kong, L. Y., B. C. Wilson, et al. (1996). "The effects of the HIV-1 envelope protein gp120 on the production of nitric oxide and proinflammatory cytokines in mixed glial cell cultures." Cell Immunol 172(1): 77-83.
Li, Y., L. Fu, et al. (2004). "Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia." J Virol 78(7): 3398-406.
Lim, J. K., J. M. Burns, et al. (2005). "Multiple pathways of amino terminal processing produce two truncated variants of RANTES/CCL5." J Leukoc Biol 78(2): 442-52.
Lin, T. Y., S. H. Hsia, et al. (2003). "Proinflammatory cytokine reactions in enterovirus 71 infections of the central nervous system." Clin Infect Dis 36(3): 269-74.
Lindenbach, B. D. and C. M. Rice (1997). "trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication." J Virol 71(12): 9608-17.
Lombardi, V. R., M. Garcia, et al. (1998). "Microglial activation induced by factor(s) contained in sera from Alzheimer-related ApoE genotypes." J Neurosci Res 54(4): 539-53.
Luo, Y., M. A. Berman, et al. (2003). "Tumor necrosis factor is required for RANTES-induced astrocyte monocyte chemoattractant protein-1 production." Glia 43(2): 119-27.
Mander, P., V. Borutaite, et al. (2005). "Nitric oxide from inflammatory-activated glia synergizes with hypoxia to induce neuronal death." J Neurosci Res 79(1-2): 208-15.
Marcus, J. S., S. L. Karackattu, et al. (2003). "Cytokine-stimulated inducible nitric oxide synthase expression in astroglia: role of Erk mitogen-activated protein kinase and NF-kappaB." Glia 41(2): 152-60.
Marques, C. P., S. Hu, et al. (2004). "Interleukin-10 attenuates production of HSV-induced inflammatory mediators by human microglia." Glia 47(4): 358-66.
Nakamura, Y., M. Ohmaki, et al. (2003). "Involvement of protein kinase C in glutamate release from cultured microglia." Brain Res 962(1-2): 122-8.
Nowak, T. and G. Wengler (1987). "Analysis of disulfides present in the membrane proteins of the West Nile flavivirus." Virology 156(1): 127-37.
Okamoto, M., X. Wang, et al. (2005). "HIV-1-infected macrophages induce astrogliosis by SDF-1alpha and matrix metalloproteinases." Biochem Biophys Res Commun 336(4): 1214-20.
Olson, J. K., A. M. Girvin, et al. (2001). "Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler''s virus." J Virol 75(20): 9780-9.
Orrenius, S., D. J. McConkey, et al. (1989). "Role of Ca2+ in toxic cell killing." Trends Pharmacol Sci 10(7): 281-5.
Park, C. H., K. Matsuda, et al. (2003). "Persistence of viral RNA segments in the central nervous system of mice after recovery from acute influenza A virus infection." Vet Microbiol 97(3-4): 259-68.
Patel, C. A., M. Mukhtar, et al. (2000). "Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells." J Virol 74(20): 9717-26.
Pawate, S., Q. Shen, et al. (2004). "Redox regulation of glial inflammatory response to lipopolysaccharide and interferongamma." J Neurosci Res 77(4): 540-51.
Persson, M., M. Brantefjord, et al. (2005). "Lipopolysaccharide increases microglial GLT-1 expression and glutamate uptake capacity in vitro by a mechanism dependent on TNF-alpha." Glia 51(2): 111-20.
Randall, R. D. and S. A. Thayer (1992). "Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons." J Neurosci 12(5): 1882-95.
Rash, J. E., T. Yasumura, et al. (2001). "Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons." J Neurosci 21(6): 1983-2000.
Ravi, V., A. S. Desai, et al. (1993). "Persistence of Japanese encephalitis virus in the human nervous system." J Med Virol 40(4): 326-9.
Reiss, C. S. and T. Komatsu (1998). "Does nitric oxide play a critical role in viral infections?" J Virol 72(6): 4547-51.
Rey, F. A., F. X. Heinz, et al. (1995). "The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution." Nature 375(6529): 291-8.
Rothman, S. M. and J. W. Olney (1986). "Glutamate and the pathophysiology of hypoxic--ischemic brain damage." Ann Neurol 19(2): 105-11.
Salinska, E., W. Danysz, et al. (2005). "The role of excitotoxicity in neurodegeneration." Folia Neuropathol 43(4): 322-39.
Sharma, S., A. Mathur, et al. (1991). "Japanese encephalitis virus latency in peripheral blood lymphocytes and recurrence of infection in children." Clin Exp Immunol 85(1): 85-9.
Slepko, N., M. Patrizio, et al. (1999). "Expression and translocation of protein kinase C isoforms in rat microglial and astroglial cultures." J Neurosci Res 57(1): 33-8.
Srinivasan, D., J. H. Yen, et al. (2004). "Cell type-specific interleukin-1beta signaling in the CNS." J Neurosci 24(29): 6482-8.
Su, C. M., C. L. Liao, et al. (2001). "Highly sulfated forms of heparin sulfate are involved in japanese encephalitis virus infection." Virology 286(1): 206-15.
Takeuchi, H., S. Jin, et al. (2006). "Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner." J Biol Chem 281(30): 21362-8.
Wang, S. M., H. Y. Lei, et al. (2003). "Pathogenesis of enterovirus 71 brainstem encephalitis in pediatric patients: roles of cytokines and cellular immune activation in patients with pulmonary edema." J Infect Dis 188(4): 564-70.
Wu, Z., Y. Hayashi, et al. (2007). "Involvement of prostaglandin E2 released from leptomeningeal cells in increased expression of transforming growth factor-beta in glial cells and cortical neurons during systemic inflammation." J Neurosci Res 85(1): 184-92.
Yang, K. D., W. T. Yeh, et al. (2004). "A model to study neurotropism and persistency of Japanese encephalitis virus infection in human neuroblastoma cells and leukocytes." J Gen Virol 85(Pt 3): 635-42.