(3.238.7.202) 您好!臺灣時間:2021/02/25 11:01
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:黃揚惠
研究生(外文):Yang-hui Huang
論文名稱:探討G-CSF在癲癇動物模式中的影響
論文名稱(外文):The effect of G-CSF in an animal model of epilepsy
指導教授:劉昭麟劉昭麟引用關係李旺祚李旺祚引用關係
指導教授(外文):Chao-lin LiuWang-tso Lee
學位類別:碩士
校院名稱:明志科技大學
系所名稱:生化工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:60
中文關鍵詞:顳葉癲癇紅藻氨酸顆粒球生長激素
外文關鍵詞:TLEKAG-CSF
相關次數:
  • 被引用被引用:0
  • 點閱點閱:181
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
顳葉癲癇(Temporal lobe epilepsy, TLE)為常見的腦部疾病之ㄧ,其不定期的發作會造成病人生活上的不便。紅藻氨酸(kainite; KA)為麩氨酸(glutamate)的類似物,常被用來誘發癲癇的動物模式,其主要導致海馬迴(hippocampus)的損傷尤其是在CA1及CA3的區域。本研究主要利用KA誘發TLE的大鼠動物模式,來探討KA刺激幹細胞增生的時間點以及顆粒球生長激素(Granulocyte colony-stimulating factor; G-CSF)對於KA所誘發TLE動物模式的治療效果及其可能的機制。
我們發現KA可造成海馬迴CA3區域的損傷,並且刺激海馬迴CA3及CA1區域的神經膠細胞增生。利用腹腔注射KA 120分鐘後有68% 的大鼠會達到第六階段的抽慉。我們同時利用5-溴2-脫氧尿嘧啶核苷(5-bromo-2’deoxyurdine; BrdU)來標定新生的幹細胞,結果發現KA所刺激的幹細胞增生能夠持續至少三天,第五天在統計上雖然無顯著增加之結果,但仍可觀察到幹細胞的增生的趨勢。另外,我們利用BrdU及膠纖維酸性蛋白(glial fibrillary acidic protein; GFAP)交叉免疫染色發現,KA所刺激產生的新生幹細胞只有4.82%分化為神經膠細胞,所以KA所造成的神經膠細胞增生並非主要由新生的幹細胞分化而來。此外,我們利用被動閃避學習系統(passive avoidance test)證實,KA會導致動物海馬迴的損傷而造成記憶能力的喪失。為了更進一步尋求治療抽慉所致海馬迴傷害的方法,我們更進一步利用G-CSF刺激TLE動物的幹細胞增生,我們發現經由G-CSF治療過後的TLE動物,其海馬迴CA1以及CA3的周邊會出現大量新生的幹細胞,並且利用被動閃避學習系統證實G-CSF的確能夠改善TLE動物的記憶能力。
由於G-CSF可刺激海馬迴CA1及CA3周邊的幹細胞增生,並改善TLE動物的記憶功能,希望在未來能夠應用於臨床上造福癲癇病人。
Background: Most of the current clinical treatments for temporal lobe epilepsy (TLE) are largely symptomatic and was usually accompanied with serious adverse effects. In the present study, we investigated the possible neurogenesis in an animal model of kainic acid (KA)-induced seizures, and searched for potential treatment for KA-induced hippocampal damage.
Methods and Results: Administration of KA, an analogue of the excitatory amino acid glutamate, can induce seizures in rats, leading to hippocampal lesions closely resembling human TLE. In the current study, we used KA to induce seizures, which resulted in hippocampal lesions, especially in CA3 region. It showed that GFAP positive cells, representative for glia cells, were also increased in CA3 and CA1 areas at one week after KA injection. Additionally, KA-induced seizures were followed by the increase of BrdU-positive cells, which persisted for at least 3 days in both hippocampal CA1 and CA3 regions. However, only 5 % of BrdU-positive cells appeared to be GFAP positive, indicating that only few proliferating stem cells were differentiated into glia cells. Passive avoidance test was used to evaluate the behavioral changes in rats, and it revealed impaired memory in KA-treated rats. When G-CSF was supplemented to these rats, the number of newborn stem cells was relatively increased in peri-CA1 and CA3 areas, and the number of apoptotic neurons in hippocampus at one week post KA administration was attenuated. Furthermore, the memory function of rats treated with G-CSF was also improved.
Conclusion: KA is able to induce neuronal death in hippocampal area and behavioral change in rats. There was also significant neurogenesis persisted for at least 3 days in hippocampal area. G-CSF therapeutic approach, which is able to attenuate the neuronal death and increase the number of stem cells in hippocampus, can be of great potential in the future to treat patients with temporal lobe epilepsy.
中文摘要 v
Abstract vii
I. Introduction 1
The Pathogenesis of Epilepsy 1
Hippocampus and Related Pathways 2
KA as One of the Animal Models of Epilepsy 3
Neural Stem Cells and Their Applications 6
G-CSF 8
II. Experiment aim 10
III. Experiment flowchart 11
IV. Experiment methods 12
4.1 animals 12
4.2 reagents preparation 12
4.2.1 8% paraformaldehyde 12
4.2.2 0.2M pasphate buffer, pH7.4 12
4.2.3 4% paraformaldehyde in 100mM phosphate buffer 12
4.2.4 BrdU 12
4.2.5 Diethypyrocarbonate (DEPC) water 12
4.2.6 antifreezer 12
4.2.7 blocking reagent 12
4.3 TLE rat model 13
4.4 Onset of KA-induced neurogenesis 13
4.5 Tissue processing 13
4.6 BrdU and Hochest/GFAP double immunohistochemistry 13
4.7 Hematoxylin and Eosin (H&E) Immunohistochemistry 14
4.8 G-CSF treatment 14
4.9 Passive avoidance test 14
V. Expriment results 16
5.1 KA induced the TLE rat model 16
5.2 Effects of KA on hippocampal neuronal degeneration 17
5.3 KA-induced cell proliferation and gliosis induced by KA 17
5.4 Cell differentiation of KA-induced new born cell 18
5.5 G-CSF stimulated stem cell mobilization and homing in the brains of TLE rat model 18
VI.Discussion 20
Seizure induced stem cell proliferation 20
Newborn stem cell differentiation 20
G-CSF therapy in brain injury animal model 21
VII.Figures 23
VIII. Tables 45
IX. Reference 48
Ahlenius, S., Oprica, M., Eriksson, C., Winblad, B. & Schultzberg, M. (2002). Effects of kainic acid on rat body temperature: unmasking by dizocilpine. Neuropharmacology, 43(1), 28-35.
Banasr, M., Hery, M., Brezun, J. M., & Daszuta, A. (2001). Serotonin mediates oestrogen stimulation of cell proliferation in the adult dentate gyrus. Eur J Neurosci, 14(9), 1417-1424.
Ben-Ari, Y. (1985). Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy. Neuroscience, 14(2), 375-403.
Ben-Ari, Y. & Cossart, R. (2000). Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci, 23(11), 580-587.
Ben-Ari, Y., Tremblay, E. & Ottersen, O. P. (1980). Injections of kainic acid into the amygdaloid complex of the rat: an electrographic, clinical and histological study in relation to the pathology of epilepsy. Neuroscience, 5(3), 515-528
Benkovic, S. A., O'Callaghan, J. P., & Miller, D. B. (2006). Regional neuropathology following kainic acid intoxication in adult and aged C57BL/6J mice. Brain Res, 1070(1), 215-231.
Bernabeu, R., & Sharp, F. R. (2000). NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus. J Cereb Blood Flow Metab, 20(12), 1669-1680.
Bodine, D. M., Seidel, N. E., Gale, M. S., Nienhuis, A. W. & Orlic, D. (1994). Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor. Blood, 84(5), 1482-1491.
Chang, Y. C., Huang, A. M., Kuo, Y. M., Wang, S. T., Chang, Y. Y., & Huang, C. C. (2003). Febrile seizures impair memory and cAMP response-element binding protein activation. Ann Neurol, 54(6), 706-718.
Coyle, J. T., Molliver, M. E., & Kuhar, M. J. (1978). In situ injection of kainic acid: a new method for selectively lesioning neural cell bodies while sparing axons of passage. J Comp Neurol, 180(2), 301-323.
Dalby, N. O. & Mody, I. (2001). The process of epileptogenesis: a pathophysiological approach. Curr Opin Neurol, 14(2), 187-192.

Dube, C., Richichi, C., Bender, R. A., Chung, G., Litt, B., & Baram, T. Z. (2006). Temporal lobe epilepsy after experimental prolonged febrile seizures: prospective analysis. Brain, 129(Pt 4), 911-922.
Engel, J., Jr. (1996). Clinical evidence for the progressive nature of epilepsy. Epilepsy Res Suppl, 12, 9-20.
Ferkany, J. W., Zaczek, R. & Coyle, J. T. (1982). Kainic acid stimulates excitatory amino acid neurotransmitter release at presynaptic receptors. Nature, 298(5876), 757-759.
Holmes, G. L., Gairsa, J. L., Chevassus-Au-Louis, N., & Ben-Ari, Y. (1998). Consequences of neonatal seizures in the rat: morphological and behavioral effects. Ann Neurol, 44(6), 845-857.
Krnjevic, K., Morris, M. E. & Reiffenstein, R. J. (1980). Changes in extracellular Ca2+ and K+ activity accompanying hippocampal discharges. Can J Physiol Pharmacol, 58(5), 579-582.
Lee, C. L., Hannay, J., Hrachovy, R., Rashid, S., Antalffy, B., & Swann, J. W. (2001). Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy. Neuroscience, 107(1), 71-84.
McKay, R. D. (1999). Brain stem cells change their identity. Nat Med, 5(3), 261-262.
Moon, C., Yoo, J. Y., Matarazzo, V., Sung, Y. K., Kim, E. J., & Ronnett, G. V. (2002). Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation. Proc Natl Acad Sci U S A, 99(13), 9015-9020.
Nadler, J. V. (1979). Kainic acid: neurophysiological and neurotoxic actions. Life Sci, 24(4), 289-299.
Nadler, J. V. (1981). Minireview. Kainic acid as a tool for the study of temporal lobe epilepsy. Life Sci, 29(20), 2031-2042.
Nadler, J. V., Perry, B. W., Gentry, C., & Cotman, C. W. (1980). Degeneration of hippocampal CA3 pyramidal cells induced by intraventricular kainic acid. J Comp Neurol, 192(2), 333-359.
Owens, D. F., & Kriegstein, A. R. (2002). Is there more to GABA than synaptic inhibition? Nat Rev Neurosci, 3(9), 715-727.


Ozawa, S., Kamiya, H. & Tsuzuki, K. (1998). Glutamate receptors in the mammalian central nervous system. Prog Neurobiol, 54(5), 581-618.
Panchision, D. M., & McKay, R. D. (2002). The control of neural stem cells by morphogenic signals. Curr Opin Genet Dev, 12(4), 478-487.
Phelps, S., Mitchell, J. & Wheal, H. V. (1991). Changes to synaptic ultrastructure in field CA1 of the rat hippocampus following intracerebroventricular injection of kainic acid. Neuroscience, 40(3), 687-699.
Pisa, M., Sanberg, P. R., Corcoran, M. E., & Fibiger, H. C. (1980). Spontaneously recurrent seizures after intracerebral injections of kainic acid in rat: a possible model of human temporal lobe epilepsy. Brain Res, 200(2), 481-487.
Popescu, B. O., Oprica, M., Sajin, M., Stanciu, C. L., Bajenaru, O., Predescu, A., Vidulescu, C. & Popescu, L. M. (2002). Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo. J Cell Mol Med, 6(4), 555-569.
Racine, R. J. (1972). Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol, 32(3), 281-294.
Sanchez, R. M., Koh, S., Rio, C., Wang, C., Lamperti, E. D., Sharma, D., et al. (2001). Decreased glutamate receptor 2 expression and enhanced epileptogenesis in immature rat hippocampus after perinatal hypoxia-induced seizures. J Neurosci, 21(20), 8154-8163.
Schwob, J. E., Fuller, T., Price, J. L., & Olney, J. W. (1980). Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study. Neuroscience, 5(6), 991-1014.
Shetty, A. K., Zaman, V., & Shetty, G. A. (2003). Hippocampal neurotrophin levels in a kainate model of temporal lobe epilepsy: a lack of correlation between brain-derived neurotrophic factor content and progression of aberrant dentate mossy fiber sprouting. J Neurochem, 87(1), 147-159.
Shingo, T., Sorokan, S. T., Shimazaki, T., & Weiss, S. (2001). Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci, 21(24), 9733-9743.
Shyu, W. C., Lin, S. Z., Yang, H. I., Tzeng, Y. S., Pang, C. Y., Yen, P. S., et al. (2004). Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells. Circulation, 110(13), 1847-1854.
Sperk, G., Lassmann, H., Baran, H., Seitelberger, F. & Hornykiewicz, O. (1985). Kainic acid-induced seizures: dose-relationship of behavioural, neurochemical and histopathological changes. Brain Res, 338(2), 289-295.
Tanaka, T., Tanaka, S., Fujita, T., Takano, K., Fukuda, H., Sako, K. & Yonemasu, Y. (1992). Experimental complex partial seizures induced by a microinjection of kainic acid into limbic structures. Prog Neurobiol, 38(3), 317-334.
Tremblay, E., Nitecka, L., Berger, M. L., & Ben-Ari, Y. (1984). Maturation of kainic acid seizure-brain damage syndrome in the rat. I. Clinical, electrographic and metabolic observations. Neuroscience, 13(4), 1051-1072.
Tsai, R. Y., & McKay, R. D. (2000). Cell contact regulates fate choice by cortical stem cells. J Neurosci, 20(10), 3725-3735.
Vicario-Abejon, C., Collin, C., Tsoulfas, P., & McKay, R. D. (2000). Hippocampal stem cells differentiate into excitatory and inhibitory neurons. Eur J Neurosci, 12(2), 677-688.
Vicario-Abejon, C., Johe, K. K., Hazel, T. G., Collazo, D., & McKay, R. D. (1995). Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons. Neuron, 15(1), 105-114.
Weaver, C. H., Buckner, C. D., Longin, K., Appelbaum, F. R., Rowley, S., Lilleby, K., Miser, J., Storb, R., Hansen, J. A. & Bensinger, W. (1993). Syngeneic transplantation with peripheral blood mononuclear cells collected after the administration of recombinant human granulocyte colony-stimulating factor. Blood, 82(7), 1981-1984.
Wexler, E., & Palmer, T. (2002). Where, oh where, have my stem cells gone? Trends Neurosci, 25(5), 225-227.
Willow, M., Gonoi, T. & Catterall, W. A. (1985). Voltage clamp analysis of the inhibitory actions of diphenylhydantoin and carbamazepine on voltage-sensitive sodium channels in neuroblastoma cells. Mol Pharmacol, 27(5), 549-558.
Willing, A. E., Lixian, J., Milliken, M., Poulos, S., Zigova, T., Song, S., et al. (2003). Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J Neurosci Res, 73(3), 296-307.
Zagrean, L., Varlas, V., Oprica, M., Munteanu, A. M., Oltenschi, C. & Voicu, T. (1993). EEG study of kainate-induced epilepsy in non-anaesthetized freely moving rats. Rom J Physiol, 30(1-2), 115-118.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔