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대뇌 허혈과 재관류 손상에서의 glutamate dehydrogenase활성에 의한 신경세포보호
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 백은주 | - |
| dc.contributor.author | 김아영 | - |
| dc.date.accessioned | 2019-10-21T07:30:09Z | - |
| dc.date.available | 2019-10-21T07:30:09Z | - |
| dc.date.issued | 2017-02 | - |
| dc.identifier.other | 24343 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/19027 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2017. 2 | - |
| dc.description.tableofcontents | ABSTRACT i TABLE OF CONTENTS iii LIST OF FIGURES vi LIST OF TABLES vii ABBREVIATION viii I. INTRODUCTION 1 II. MATERIALS AND METHODS 8 1. FOCAL CEREBRAL ISCHEMIA AND REPERFUSION 8 2. ASSESSMENT OF CELL DEATH 8 3. CELL CULTURES 9 3.1. Cortical neurons 9 3.2. Cortical neurons and glias co-culture 10 3.3. Astrocytes 10 4. IN VITRO STROKE INDUCTION 10 4.1. Oxygen and glucose depletion (OGD) 10 4.2. Iodoacetate (IOA) treatment 11 5. MTT CONVERSION ASSAY 11 6. MEASUREMENT OF ROS 11 7. MEASUREMENT OF TOTAL THIOLS 12 8. IN VITRO ANTIOXIDANT ACTIVITY ASSAYS 12 8.1. DPPH radical scavenging activity 12 8.2. Metal chelating activity 12 8.3. Reducing power activity 13 9. IMMUNOCYTOCHEMISTRY 13 10. CALCEIN-AM AND PROPIDIUM IODIDE STAINING 14 11. TISSUE PREPARATION 14 12. MEASUREMENT OF ATP 14 13. MEASUREMENT OF PYRUVATE, L- and D-LACTATE, AND α-KETOGLUTARATE 15 14. ENZYME ACTIVITY ASSAYS 15 14.1. GAPDH ACTIVITY 15 14.2. GDH ACTIVITY 16 14.3. AAT ACTIVITY 17 14.4. PAG ACTIVITY 17 15. WESTERN BLOTTING AND ANALYSIS 17 16. AMINO ACID ANALYSIS 19 17. STATISTICAL ANALYSIS 20 III. RESUTLS 21 A. β-Lapachone (βLA) alleviated neuronal damage against cerebral ischemia and reperfusion (I/R) 21 B. βLA alleviated neuronal damage against BSO or IOA 25 C. βLA protected cultured neurons from oxidative stress 28 D. βLA did not have structural antioxidant activity 31 E. βLA alleviated damage of cultured brain cells from IOA injury 34 F. βLA restored intracellular ATP in in vivo and in vitro stroke induction 37 G. βLA assisted in the protective effect of pyruvate 40 H. βLA accelerated the use of glutamate as an alternative energy source 42 I. βLA directly increased GDH activity 45 J. GDH activation supplied the energy required to protect from energy depletion 47 K. GDH activation by BCH supplied the energy required to protect neurons with energy failure 50 L. βLA-mediated glutamate metabolism was not involved in AAT activity 52 M. Glutamine catabolism by PAG maintained glutamate supplement 54 N. Glutamate supplement by PAG maintained neuronal survival 57 O. Impaired glucose metabolism affected the intracellular amino acid content 59 P. βLA relieved methylglyoxal-induced neuronal toxicity. 62 Q. βLA reduced the accumulation of intracellular D-lactate 66 R. MG-induced neuronal death was not involved in energy failure 69 S. βLA improved the intracellular GSH levels 71 IV. DISCUSSION 73 V. REFERENCES 81 국문초록 94 | - |
| dc.language.iso | kor | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 대뇌 허혈과 재관류 손상에서의 glutamate dehydrogenase활성에 의한 신경세포보호 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2017. 2 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 770697 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000024343 | - |
| dc.subject.keyword | neuroprotection | - |
| dc.subject.keyword | focal ischemia | - |
| dc.subject.keyword | reperfusion | - |
| dc.subject.keyword | energy metabolism | - |
| dc.subject.keyword | glutamate dehydrogenase | - |
| dc.description.alternativeAbstract | Energy metabolism in the brain is important during normal function and in pathological conditions, especially stroke. Although glucose is a main obligatory substrate, the brain can use other energy substrates, including monocarboxylic acid, ketone bodies, amino acids, and fatty acids during glucose restriction. In particular, glutamate is the most abundant excitatory amino acid, and deregulation of glutamate homeostasis is associated with degenerative neurological disorders. Glutamate dehydrogenase (GDH) is important for glutamate metabolism and plays a central role in expanding the pool of tricarboxylic acid cycle intermediate alpha-ketoglutarate (α-KG), which improves overall bioenergetics. Under high energy demand, maintenance of ATP production results in functionally active mitochondria. Here, it is examined whether the modulation of GDH activity can rescue ischemia/reperfusion-induced neuronal death in an in vivo mouse model of middle artery occlusion and an in vitro oxygen/glucose depletion model. Iodoacetate, an inhibitor of glycolysis, was also used in a model of energy failure, remarkably depleting ATP and α-KG. To stimulate GDH activity, the GDH activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid and potential activator beta-lapachone were used. The GDH activators restored α-KG and ATP levels in the injury models and provided potent neuroprotection. It was also found that beta-lapachone increased glutamate utilization, accompanied by a reduction in extracellular glutamate. In addition, the glutamate consumed by beta-lapachone was supplied from glutamine with phosphate-activated glutaminase (PAG) reaction. Thus, the hypothesis that mitochondrial GDH activators increase α-KG production as an alternative energy source for use in the tricarboxylic acid cycle under energy-depleted conditions was confirmed. The results suggest that increasing GDH-mediated glutamate oxidation represents a new therapeutic intervention for neurodegenerative disorders, including stoke. | - |
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