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뇌외상 동물모델에서 인간 골수유래 중간엽줄기세포의 치료효과에 관한 연구: 신경성장인자의 분비와 세포사멸 억제
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 김세혁 | - |
| dc.contributor.author | 김화정 | - |
| dc.date.accessioned | 2019-10-21T07:14:11Z | - |
| dc.date.available | 2019-10-21T07:14:11Z | - |
| dc.date.issued | 2009-02 | - |
| dc.identifier.other | 9585 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/17595 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의학과,2009. 2 | - |
| dc.description.tableofcontents | ABSTRACT i TABLE OF CONTENTS iii LIST OF FIGURES vi LIST OF TABLES ix ABBREVIATION x I. INTRODUCTION 1 II. MATERIALS AND METHODS 4 1. Animal model of TBI 4 2. Impact velocity calibration 4 3. Assessment of neurological function 5 4. Optical microscopic observation of preliminary laboratory animals 7 5. Impact velocity and depth of deformation 7 6. Measurement of injured area 7 7. Isolation and preparation of hMSCs for transplantation 8 8. Experimental groups 9 9. Enzyme-Linked Immunosorbent Assay (ELISA) 13 10. Immunohistochemical evaluation 13 11. Western blot analysis and immunoprecipitation 14 11-1. Western blot analysis 14 11-2. Immunoprecipitation 14 11-3. pAkt expression in neurotrophins-treated PC12 cells 15 12. Determination of NGF, NT-3 and BDNF mRNA levels 16 12-1. RNA extraction 16 12-2. Reverse transcription (RT) 16 12-3. PCR 17 13. Statistical Analysis 19 III. RESULT 20 Part A. Animal Model of Traumatic Brain Injury Induced by Controlled Cortical Impact Device 20 1. Impact velocity with differential compressed air pressure 20 2. Changes of behavioral score after injury 20 3. Optical microscopic observation 20 4. Changes in the neurobehavioural scores according to the different impact velocity and the different depth of deformation 21 4-1. Rotarod motor test 21 4-2. Modified neurological severity score 21 5. Morphological changes (TTC stain result) 22 Part B. Therapeutic effects of human mesenchymal stem cells on traumatic brain injury in rats 29 1. Neurological function improved significantly in rats with TBI that received hMSCs treatment 29 2. Transplanted hMSCs migrated to perilesional area 29 3. Minor portion of transplanted hMSCs differentiated into neuronal or glial cells 31 4. Tissue levels of NGF, NT-3 and BDNF were significantly increased in the hMSCs-treated group 35 5. A little increased concentration of NGF in hMSCs cultures medium 37 6. Transplanted hMSCs migrated to the perilesional area and secreted growth factors 39 7. Relative expression of neurotrophin mRNA in traumatized rat brain 41 8. Detection of growth factors in protein level (western blot and Immunoprecipitation) 41 9. Detection of receptor on growth factors 44 10. Up-regulation of pAkt and reduced caspase-3 cleavage in the hMSCs-treated group 47 IV. DISCUSSION 52 Part A. Animal Model of Traumatic Brain Injury Induced by Controlled Cortical Impact Device 52 Part B. Therapeutic effects of human mesenchymal stem cells on traumatic brain injury in rats 55 V. CONCLUSION 59 REFERENCES 61 국문요약 70 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 뇌외상 동물모델에서 인간 골수유래 중간엽줄기세포의 치료효과에 관한 연구: 신경성장인자의 분비와 세포사멸 억제 | - |
| dc.title.alternative | Kim hwa jung | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Kim hwa jung | - |
| dc.contributor.department | 일반대학원 의학과 | - |
| dc.date.awarded | 2009. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 567723 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000009585 | - |
| dc.subject.keyword | traumatic brain injury | - |
| dc.subject.keyword | human mesenchymal stem cells | - |
| dc.subject.keyword | neurotrophic factors | - |
| dc.subject.keyword | apoptosis | - |
| dc.subject.keyword | functional recovery | - |
| dc.description.alternativeAbstract | Traumatic brain injury (TBI) leads to secondary brain insults, which often result in permanent functional loss of damaged cells. Many researchers have looked for methods that will reduce secondary brain damage since there is no effective therapeutics to recover functional loss of damaged tissues. The severity of the secondary mechanism of TBI depends upon two factors: injury severity (mild, moderate or severe) and location of the primary injury. Secondary brain damage has been known to involve neuroinflammatory, apoptotic and oxidative stress mechanisms which are mainly dependent on intracerebral production of cytokines. The development of an experimental model that resembles pathological and functional changes in human brain is critical in providing an effective therapeutics for human TBI. The controlled cortical impact (CCI) model is frequently used for TBI models in animals because it enables the physical adjustment of injury levels and has close pathological resemblance to actual human TBI. However, little is known about the severity of brain injury according to injury-induction parameters. In this study, morphological changes and behavioral scores after TBI in rats were measured according to the different impact velocity and depth of deformation; these parameters were further investigated to provide a guideline for establishing a TBI animal model. In recent years, there has been increasing interest in the use of mesenchymal stem cells (MSCs) as cell therapy for TBI. To investigate the therapeutic effects and their possible mechanisms of human bone marrow-derived mesenchymal stem cells (hMSCs) in the rat model of TBI, we analyzed the neurological functions and in vivo growth factor production. Male Sprague-Dawley rats were injured with a CCI device and divided into two experimental groups: those which received hMSCs intravenously 24 hours after TBI and those injected with saline only as placebo. For the analysis of in vivo production of growth factors, the animals were sacrificed 2, 8, 15, and 29 days (n=6 for each group at each day) after TBI, and the injured hemispheres were extracted. We measured the level of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the brain, using enzyme-linked immunosorbent assay (ELISA). The neurological function assessed by rotarod motor test and modified neurological severity scores (mNSS) recovered significantly in the animals treated with hMSCs 15 days after TBI. Quantitative ELISA studies showed that the in vivo expressions of NT-3 and NGF 2 and 8 days after TBI and the in vivo expression of BDNF 2 days after TBI were significantly increased (p < 0.05) in the treated group compared to the placebo group. Additionally, NGF and BDNF mRNA expression were up-regulated 2 and 8 days after TBI in the animals treated with hMSCs. Immunohistochemical studies showed that the expression of NGF, BDNF, and NT-3 were stronger in the injured hemispheres of the treated group compared to those of the placebo group 2 days after TBI. Western blotting showed that pAkt expression was up-regulated 2 days after TBI and caspase-3 cleavage was significantly decreased 8 days after TBI in hMSCs group. Our results suggest that treatment of TBI with hMSCs during the acute phase can enhance the functional outcome and the augmentation of neurotrophic factors in the injured hemisphere can be one of possible mechanisms for functional recovery by reducing neuronal apoptosis. | - |
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