臺灣博碩士論文加值系統

中文摘要
長久以來，人們認為中樞神經系統(central nervus system)是一個免疫特權區(immunologically privileged site) ，主要是因為中樞神經系統缺乏淋巴組織，且其血管外層還包圍了一層由星狀細胞(astrocyte)所組成的血腦障壁(blood-brain barrier, BBB)，以及FasL(CD95L) 及TGFβ (transformation factor-β) 的表現所致。但在過去的研究中發現，週邊給予LPS (lipopolysaccharide)刺激後所產生的細胞激素(cytokine) 如TNFα (tumor necrosis factor-α)、IL-1β(interleukin-1β)、IL-6 (interleukin-6)等，可活化下視丘-腦垂體-腎上腺軸(hypothalamic-pituitary-adrenocortical axis, HPA axis)，進而引起生理現象的改變如發燒。這些結果顯示出，中樞神經系統與週邊免疫系統間的交流是存在的。一般認為腦部因為有血腦障壁的存在，所以週邊發炎所產生的細胞激素是不會進入到腦部的，於是細胞激素是如何影響中樞神經系統的疑問就產生了。在我們的研究中，我們利用完全佛蘭氏佐劑(complete Freund’s adjuvant，CFA)或完全佛蘭氏佐劑混poly(Glu60 Ala30 Tyr10 , GAT )給予老鼠週邊刺激，引起鼠腦表現細胞激素。以免疫化學染色的方法我們發現原本在腦中不表現的TNFα及COX-2 (cyclooxygenase type 2)，於完全佛蘭氏佐劑或完全佛蘭氏佐劑混和GAT刺激後可表現在微血管上，也發現IL-1β只表現在免疫完全佛蘭氏佐劑混和GAT的鼠腦中。相反的原本表現在腦部神經束細胞的IL-1α及ependymal cell的IL-6經此刺激後卻會下降。利用半定量的反轉錄-聚合連鎖反應證明了腦部有自行產生細胞激素的能力。我們亦利用會持續表現β-galactosidase 的M4 E. coli 當作偵測血腦障壁通透性的工具，結果發現在免疫完全佛蘭氏佐劑混和GAT的老鼠中，其血腦障壁通透性最大的時間點是48小時；而免疫完全佛蘭氏佐劑的老鼠，其血腦障壁通透性最大的時間點是96小時。且血腦障壁通透性增加的現象可被NS398 (COX-2抑制劑)及TNFα的抗血清所抑制。另外也由免疫化學染色發現在處理過NS398後，鼠腦中的TNFα表現量會降低；而在處理過TNFα抗血清後鼠腦的COX-2表現量會降低；我們也發現在免疫完全佛蘭氏佐劑混和GAT的老鼠在48小時時會產生發燒現象，此發燒的現象可被NS398或是indomethacin (COX-1及COX-2抑制劑)所抑制，但卻不能被TNFα的抗血清所抑制，顯示出cyclooxygenase 可參與發燒反應。綜合上述結果，我們認為週邊的發炎反應除了可引起腦部微血管細胞表現TNFα、IL-1β之外，在微血管的TNFα可促使COX-2的產生，進而導致血腦障壁通透性的增加，而IL-1β也透過COX-2的作用引起發燒反應。

英文摘要
The CNS (central nervous system) of mammal is considered to be an immunologically privileged site because it lacks lymphatic drainage, the brain is separated from the blood compartment by the blood-brain barrier (BBB). The brain also expresses FasL and TGFβ that can down-modulate the immune response. However, peripheral administration of lipopolysaccharide has been found to stimulate the hypothalamic- pituitary- adrenocortical axis to induce fever and influence behavior. This result showed that communication between peripheral immune system and CNS exists. It is general believed that cytokines produced in peripheral tissue can not cross the BBB because of the unique features of brain microvascular endothelial cells that wrapped by astrocyte. In this study, we reported that the peripheral stimulation with complete Freund’s adjuvant (CFA) induced the cytokine expression in brain. With immunohistochemical staining, TNFα and cyclooxygenase type 2 (COX-2) were detected on brain microvascular vessels post CFA or poly (Glu60 Ala30 Tyr10 , GAT ) in CFA injection. IL-1βwas detected in microvascular vessels only in mice injected with GAT/CFA. On the contrary, the constitutive expression of IL-6 on brain microvascular vessels and IL-1α on brain neuron bundle were found to decrease. The cytokine induction was also confirmed by semiquantitative reverse-transcription polymerase chain reaction, indicating its de novo synthesis. Using the M4 E. coli that constitutive expresses β-galactosidaseas as a tracer to quantitate the increased permeability of BBB, we found that the vasopermeability of BBB increased maximum at 96 h post CFA injection and 48 h post GAT/CFA injection. The increase of BBB permeability was inhibited by either NS398 (COX-2 inhibitor) or anti-TNFα antiserum. Using immunohistochemistry staining, the TNFαexpression was inhibited by NS398 treatment while COX-2 induction was blocked by anti-TNFαadministration. Furthermore, peripheral administration of GAT/CFA were found to induce fever at 48 h. The fever was not inhibited by anti-TNFαantiserum, but could be inhibited by NS398 or indomethacin (COX-1 and COX-2 inhibitor). Based on the above data, we conclude that peripheral inflammation stimulates TNFα or IL-1βexpression on brain microvascular vessels. The TNFαwould further cause COX-2 induction that involved in the increase of BBB permeability. The IL-1βinduction might be responsible for the fever that can be inhibited by COX inhibitor.

目錄 頁次
中文摘要 Ⅰ
英文摘要 Ⅲ
誌謝 Ⅴ
目錄 Ⅵ
圖目錄 Ⅸ
緒論 1
材料與方法 5
Ⅰ方法 5
A.實驗動物 5
B.以週邊發炎反應誘發鼠腦表現細胞激素 5
C.藥劑之給予 5
D.老鼠體溫的測定 7
E.組織切片的備製 7
F.免疫組織化學染色(immonohistochemistry) 7
G.以持續表現β-galactosidase之突變大腸桿菌測定 8
血腦障壁（blood-brain barrier）的通透性
H.反轉錄-聚合連鎖反應 9
Ⅱ器材與試劑 13
A.儀器設備 13
B.藥品 15
結果 18
以免疫化學染色觀察腦部細胞激素的表現 18
以反轉錄聚合連鎖反應觀察腦部細胞激素的表現 19
週邊發炎反應引起血腦障壁通透性的增加 20
週邊發炎反應所引起血腦障壁通透性的增加與劑量有關而 21
與注射部位無關
抑制COX-2的產生可抑制血腦障壁通透性的增加 21
抑制TNFα的產生可抑制血腦障壁通透性的增加 22
免疫老鼠完全佛蘭氏佐劑混和GAT會引起老鼠體溫上升 23
抑制TNFα並不能抑制免疫完全佛蘭氏佐劑混和GAT老鼠 23
之發燒反應
抑制COX-2可抑制免疫完全佛蘭氏佐劑混和GAT老鼠 23
之發燒反應
討論 25
Reference 32
作者簡歷 38
著作權聲明
圖表

References:
1. Merrill, E. Jean and Etty N. Benveniste. Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci. 19:331,1998.
2. Xiao, B.G. and Link H. Immuneregulation within the central nervous system. J. Neurol. Sci.157(1):1,1998.
3. Fabry ,Z., Cedric S., Raine and Michael N. Hart. Nervous tissue as an immune compartment: the dialet of the immune response in the CNS. Immonol. Today. 15(5):218, 1994.
4. Cserr, f. Helen and Paul M. Knopf. Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain : a new view. Immunol. Today. 13(12): 507, 1992.
5. Gordon LB., Nolan Sc., Ksander BR., Knopf PM. and Harling-Berg CJ.. Normal cerebrospinal fluid suppresses the in vitro development of cytotoxic T cell: role of the brain microenviroment in CNS immune regulation. J. Neuroimmunol. 88(1-2):77,1998.
6. Tayler , A.W.and JW. Streilein. Inhibition of antigen-stimulated effector T cells by human cerebrospinal fluid. Neuroimmunomodulation. 3(2-3): 112, 1996.
7. Saas, P., Paul R. Walker, Michael hahne, Anne-Lise Quiquerez, Valerie Schnuriger Meir, Nicolas de Tribolet, Jurg Jschoppand Pierre-Yves Dietrich. Fas ligand expression by astrocytema in vivo: maintaining immune privilege in the brain ? J.Clin. Invest. 99(6):1173,1997.
8. Saper,C.B., C.D. Breder. The neurulogic basics of fever. New Engl. J. Med. 330:1880, 1994.
9. Bucala, R.. Neuruimmunomodulation by macrophage migration inhibitory factor (MIF). Ann. N.Y. Acad. Sci. 840:79,1998.
10. Dunn, A.J.and Artur H. Swiergiel. The role of cytokines in infection-related behavior. Ann. N.Y. Acad. Sci. 840:577, 1998.
11. Dantzer, R., Rose-Marie Bluthe, Sophie Laye, Jean-Luc Bret-Dibat, Patricia Parnet and Keith W. Kelley. Cytokines and sickness behavior. Ann. N.Y. Acad. Sci. 840:586,1998.
12. Pitossi, F., Adriana del Rey, Alexa Kabiersch and Hugo Besedovsky. Induction of cytokine transcripts in the central nervous system and pituitary following peripheral administration of endotoxin to mice. J. Neurosci. Res. 48:287, 1997.
13. Muller N. and Manfred Ackenheil. Psychoneuroimmunology and the cytokine action in the CNS: implications for psychiatric disorders. Prog. Neuro-Psychopharmacol.& Biol. Psychiat. 22:1,1998.
14. Rothwell, N.J.. Neuroimmune interaction: the role of cytokines. Bri. J. Pharcol. 121:841,1997.
15. Reiber, H., A.J. Sckling and M.G. Rumsby. The effect of Freund’s adjunants on blood-cerebrospinal fluid barrier permeability. J. Neurol. Sci. 63:55, 1984.
16. Farkas, G., Janson Marton, Zsusanna Nagy, Yvette Mandi, Tamas Takacs, Maria A. Dele, Csongor S. Abraham. Experimental acute pancreatitis results in increased blood-brain barrier permeability in the rat: a potential role for tumor necrosis factor and interleukin 6. Neurosci. Let. 242:147, 1998.
17. Kim, K.S., Carol A. Wass and Alan S. Cross. Blood-brain barrier permeability during the development of experimental bacterial menigitis in the rat. Experimental Nerology. 145:253, 1997.
18. Pan, Weihong, William A. Banks and Abba J. Kastin. Blood-brain barrier permeability to Ebiratide and TNF in acute spinal cord injury. Experimental Neurology. 146:367, 1997.
19. Abraham,.Csongor S., Maria A. Deli, Ferenc Joo, Pal Megyeri and Gerard Torpier. Intracarotid tumor necrosis factor-αadminitration increases the blood-brain barrier permeability in cerebral cortex of the newborn pig: quantitative aspects of double-labeling studies and confocal laser scanning analysis. Neurosci. Let. 208:85, 1996.
20. Gloor, Sergio M., Adrien Weber, Naoto Adachi and Karl Frei. Interleukin-1 modulates Protein Tyrosin phosphatase activity and permeability of brain endothelial cells. Bioche. Biophy. Res. Commun. 239:804,1997.
21. Oh, Jae-Wook, Nicholas J. Van Wagoner, Stefan Rose-John and Etty N. Benveniste. Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 Gene Expression. J. immunol. 161:4992, 1998.
22. Quan, N., M. Whiteside and M. Herkenham. Time course and localization patterns of interleukin-1β messenger RNA expression in brain and pituitary after peripheral administration of lipopolysaccharide. Neurosci. 83(1): 281, 1998.
23. Woolf, C.J., A. Allchorne, B. Safieh-Garabedian and S. Poole. Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumour necrosis factor α. Bri. J. Pharmacol. 121:417, 1997.
24. Cao, Chunyu, Kiyoshi Matsumura, Kanato Yamagata and Yasuyoshi Watanabe. Involvement of cyclooxygenase-2 in LPS induced fever and regulation of its mRNA by LPS in rat brain. Am. J. Physiol. 272:R1712, 1997.
25. Vries, Helga E. de, Margret C.M. Blom-Roosemalen, Marijke van Oosten, Albert G. de Boer, Theo J.C. van Berkel, Douwe D. Bermer and Johan Kuiper. The influuence of cytokines on the integrity of the blood brain barrier in vitro. J. Neuroimmunol. 64:37, 1996.
26. Dam, A.-M. Van, H. E. De Vries, J. Kuiper, F. J. Zijlstra, A. G. De boer, F. J. H. Tilders and F. Berkenbosch. Inteleukin—1 receptors on rat brain endothelial cells: a role in neuroimmune interaction? FASEB J. 10:351, 1996.
27. Watkins, Linda R., Steven F. Maier and Lisa E. Goehler. Cytokine-to brain communication: a review and analysis of alternative mechanisms. Life Science. 57(11): 1101, 1995.
28. Tsao, Nina, Hui-Ping Hsu and Huan-Yao Lei. TNFα-induced cyclooxyganase 2 not only increases the vasopermeability of blood-brain barrier but also enhances the neutrophil survival in Escherichia coli-induced brain inflammation. Prostaglandins & other Lipid Mediators. 57:22, 1999.
29. Milligan, E.D., Molly M. McGorry and Steven F. Maier.
Subdiaphragmatic vagotomy does not prevent fever following intracerebroventricular prostaglandin E2: further evidence for the importance of vagal afferents in immune-to-brain communication. Brain Res. 766: 240, 1997.
30. Weigent, D.A. and J. Edwin Blalock. Associations between the neuroendocrine and immune systems. J. Leukoc. Biol. 57:137, 1995.
31. Goetzl, E.J. and Sunil P. Sreedharan. Mediators of communication and adaptation in the neuroendocrine and immune systems. FASEB J. 6:2646, 1992.
32. Mucke, Lennart and Michael Eddlesion. Astrocytes in infectious and immune-mediated diseases of the central nervous system. FASEB J. 7:1226, 1993.
33. Herencia, F., A. Ubeda, M. L. Ferrandiz, M. C. Terencio, M. J. Alcaraz, R. Capaccioni and M. Paya. Anti-inflammatory activity in mice of extracts from mediterranean murine invertbrates. Life Science. 62(9):PL115, 1998.
34. Marquette, Christel, Anne-Marie Van Dam, Pierre-Emmanuel Ceccaldi, France Haour and Henri Tsiang. Induction of immunoreactive interleukin-1βand tumor necrosis factor-α in the rabies virus infected rats. J. Neuroimmunol. 68:45, 1996.
35. Weiss, J.M., Sherry A. Downie, William D. Lyman and Joan W. Berman. Astrocyte-derived Monocyte-chemoattractant protein-1 directs the transmigration of Leukocytes across a model of the human blood-brain barrier. J. Immunol. 161:6896, 1998.
36. Cho, Hee-Jung, Sang-Yul Kim, Mae-Ja Park, Dong-Sun Kim and Mi-Young Chu. Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation. Brain Res. 749:358, 1997,
37. Worrall, N.K., Kathy Chang, Wanda S. Lejeune, Thomas P. Misko and Joseph R. Williamson. TNFα causes reversible in vivo systemic vascular barrier dysfunction via NO-dependent and —independent mechanisms. Am. J. Physiol. 273:H2565, 1997.
38. Anthony, D.C., S. J. Bolton, S. Fearn and V.H. Perry. Age-related effects of IL-1β on polymorphonuclear neutrophil-dependent increases in blood-brain barrier permeability in rats. Brain. 120:435, 1997.
39. Turnbull, A.D., Soon Lee and Catherine Rivier. Mechanisms of hypothalamic-pituitary-Adrenal axis stimilation by immune signals in the adult rats. Ann. N.Y. Acad. Sci. 840:434, 1998.
40. Maier, Steven F., Lisa E. Goehler, Monika Kleshner and Linda R. Watkins. The role of the vagus nerve in cytokine-to-brain communication. Ann. N.Y. Acad. Sci. 840:289, 1998.