근위축성측삭경화증(ALS)에 대한 치료전략연구

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dc.contributor.advisor서해영-
dc.contributor.authorChoi, Chan-Il-
dc.date.accessioned2019-10-21T07:13:42Z-
dc.date.available2019-10-21T07:13:42Z-
dc.date.issued2010-02-
dc.identifier.other10520-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/17482-
dc.description학위논문(박사)--아주대학교 일반대학원 :의학과,2010. 2-
dc.description.abstract근위축성측삭경화증(Amyotrophic Lateral Sclerosis, ALS)은 운동신경세포가 선택적으로 파괴되는 퇴행성 뇌질환으로, 발병 후 운동신경세포가 점차 소실되면서 근력 약화와 근위축을 초래하여 언어장애, 사지위약, 급격한 체중감소, 폐렴 등의 증세를 보이다가 결국 3-5년 내에 호흡장애 등으로 사망하는 치명적인 질병이다. ALS의 약 10%는 가족 내 일정한 유전성을 보이지만 (Familial ALS, FALS), 대부분에서는 산발성으로 발병된다. FALS환자의 20%가 Superoxide Dismutase 1 (SOD1) 유전자 돌연변이로 발생하고 있음이 밝혀졌고, 이를 이용한 ALS 동물 모델이 개발되면서 새로운 치료제 연구가 활발히 진행되고 있다. ALS의 정확한 발병원인은 다른 여러 퇴행성 뇌질환에서와 같이 아직 명확하지 않아 특별한 치료제가 없는 상태이기 때문에, 줄기세포 치료제 개발이 크게 이슈화 되고 있다. 이번 연구에서 우리는 중간엽줄기세포 (mesenchymal stem cells, MSCs)를 신경세포로 유도하였을때, 그 치료효과가 증진될 수 있는지에 대해 연구하였다. 우리는 MSCs에 뉴로제닌1 (Neurogenin1, Ngn1)을 발현시킴으로써 신경세포로의 분화를 유도하였다. 생후 8주된 ALS 모델 생쥐를 사용하여 꼬리 정맥을 통해 106개의 신경세포로 분화 유도된 MSCs (MSCs-Ngn1)를 이식하였다. 꼬리 정맥을 통하여 이식한 MSCs-Ngn1은 이식 후 2주 후에 확인한 결과 뇌와 척수로 이동하였음이 확인되었다. 또한 MSCs-Ngn1을 이식 받은 ALS 모델 생쥐는 행동학적인 개선 효과 및 수명 연장 효과를 나타내었다. 조직학적인 분석 결과 척수 내 운동신경세포의 수에서도 MSCs-Ngn1 그룹은 차이를 보였다.. Glial cell line-derived neurotrophic factor (GDNF)는 세포의 분열과 분화 뿐만 아니라, 척수 운동신경세포 보호에 도움을 주는 신경보호 영양인자이기 때문에, ALS 치료제 개발에 있어 좋은 후보 물질로 알려져 있다. 우리는 아데노바이러스를 이용하여 GDNF를 과량 분비하는 MSCs (MSCs-GDNF)를 생산하여 ALS 모델 생쥐에 이식하고, 그 치료 효과를 확인하였다. 106개의 MSCs-GDNF를 생후 13주, 15주에 걸쳐 두차례 꼬리 정맥으로 이식한 결과, 척수 운동신경세포의 보호 및 행동학적인 개선 효과, 수명 연장 효과등을 관찰 할 수 있었다. 한편, ALS 통계조사를 통해 초기에는 남성이 여성보다 높은 발병율을 보이지만 50세 이후로는 성별차이가 사라짐을 확인하고, 이것이 여성호르몬에 의한 것인지 확인하는 실험을 진행하였다. 운동학적인 기능과 수명등을 분석한 결과, ALS 모델 수컷쥐는 암컷쥐에 비해 일주일정도 빠른 진행을 보였다. 또한 ALS 모델 암컷쥐에게 자궁 절제술을 시행하면 수컷과 같이 빠른 진행을 보이며 수명이 약7일정도 감소되었고, 자궁절제후 에스트로겐을 대체해 주면 이러한 현상이 회복됨을 확인하였다. 이를 통해 여성호르몬의 하나인 에스트로겐이 ALS 발병과 진행에 있어 영향을 준다는 것을 확인하였으며, 이러한 결과는 폐경이후 여성ALS환자의 치료 전략에 대한 새로운 시각을 제시하고 있다.-
dc.description.tableofcontentsABSTRACT ⅰ TABLE OF CONTENTS ⅲ LIST OF TABLES ⅵ LIST OF FIGURES ⅶ Ⅰ. INTRODUCTION 1 Ⅱ. MATERALS AND METHODS 6 A. Materials 6 B. Methods 7 1. Common Methods 7 1.1. Animals 7 1.2. Evaluation of motor functions 7 1.3. Assessment of disease onset and lifespan 9 1.4. Statistical analysis 9 2. (Part1) 10 2.1. Preparation of MSCs-Ngn1 cells and in vitro differentiation 10 2.2. Transplantation 10 2.3. Histological analysis of transplanted cells 11 2.4. Quantification of motor neurons and Iba1+ microglia 12 3. (Part2) 12 3.1. Western blot analysis for GDNF 13 3.2. Immunocytochemistry 13 3.3. Transplantation 14 3.4. Motor neuron counting 14 4. (Part3) 14 4.1. Ovariectomy and estrogen replacement 14 4.2. Measurement of E2 concentration and uterine weight 15 Ⅲ. RESULTS 16 A. (Part1) 16 1. In vitro cultivation of MSCs-Ngn1 16 2. Distribution of Transplanted MSCs-Ngn1 in the Central Nervous System 16 3. Protection of Spinal Motor Neurons 21 4. Immune modulation by transplanted cells 24 5. Delayed disease progression by MSCs-Ngn1 24 6. Enhanced therapeutic effects of MSCs-Ngn1 by repeated grafting 28 B. (Part2) 35 1. Evaluation of cytotoxicity and expression efficiency of adeno-GDNF virus 35 2. Effect of MSCs-GDNF administration on motor neuron survival and disease progression 38 C. (Part3) 42 1. Gender differences in onset and disease progression 42 2. Effects of female hormones in female hSOD1G93A mice 46 Ⅳ. DISCUSSION 51 Ⅴ. CONCLUSIONS 61 REFERENCES 63 국문요약 78-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title근위축성측삭경화증(ALS)에 대한 치료전략연구-
dc.title.alternativeNew Therapeutic Strategies for Amyotrophic Lateral Sclerosis Treatment-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameChan-Il Choi-
dc.contributor.department일반대학원 의학과-
dc.date.awarded2010. 2-
dc.description.degreeMaster-
dc.identifier.localId568550-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000010520-
dc.subject.keywordMesenchymal Stem Cells (MSCs)-
dc.subject.keywordAmyotrophic Lateral Sclerosis (ALS)-
dc.subject.keywordSOD1G93A mouse-
dc.subject.keywordNeurogenin1 (Ngn1)-
dc.subject.keywordGlial cell line-Derived Neurotrophic Factor (GDNF)-
dc.subject.keywordestrogen-
dc.description.alternativeAbstractAmyotrophic lateral sclerosis (ALS) is characterized by progressive dysfunction and degeneration of motor neurons in cerebral cortex, brain stem and spinal cord. Degeneration of motor neurons causes muscle weakness and respiratory paralysis, leading to decease of ALS patients. Approximately 10% of the cases are familial and 20% of those are dominantly inherited mutations in superoxide dismutase 1 (SOD1). The transgenic animal model of ALS was the G93A mouse that expresses a mutant form of human SOD1 carrying the Gly93 → Ala and develops motor neuron disease similar to familial ALS. With the lack of effective drug treatments for ALS and compelling preclinical data, new therapeutic strategies have highlighted this disease as a candidate for stem cell treatment. Here we investigated whether neural induction enhances therapeutic potentials of mesenchymal stem cells (MSCs). We induced neural induction of MSCs by overexpressing Neurogenin1, a proneural transcription factor. Depending on transplantation time, systemic transplantation of Ngn1-expressing MSCs (MSCs-Ngn1) delayed the onset of neurobehavioral degeneration or dramatically improved motor functions compared to parental MSCs in mice carrying a high copy number of a human mutant SOD1(G93A) transgene. We found that compared to MSCs, more MSCs-Ngn1 were found in hippocampus, brainstem, and the spinal cord, where most of them remained undifferentiated. We also found that more motor neurons were preserved in the spinal cord of animals with MSCs-Ngn1, suggesting that MSCs-Ngn1 migrate to the central nervous system and exert beneficial effects on host neural cells through paracrine signaling for extended period of time. Our findings strongly suggest potential benefits of neural induction of MSCs to develop successful stem cell therapy for the treatment of ALS. Glial cell line-derived neurotrophic factor (GDNF), which has been demonstrated to be the most potent neurotrophic factor for the proliferation, differentiation, and survival of spinal motor neurons, exhibits very good therapeutic potential for ALS. MSCs were infected with recombinant adenovirus GDNF (MSCs-GDNF). The MSCs-GDNF were transplanted into tail vein of ALS mice (13, 15 weeks of age), and the animals were evaluated on health and behavioral measures. Transplantation of the MSC-GDNF into tail vein delayed the onset of motor behavioral symptoms, and prolongs the life span. The incidence of ALS is higher in men than women, but the female advantage disappears with increased age. Here, we report evidence that the female advantage is due to the protective role of estrogen. In an ALS mouse model carrying the human Cu/Zn superoxide dismutase (hSOD1) G93A transgene, ovariectomy did not alter the onset age of the disease while reducing the female lifespan by 7 days and making it comparable to that of the male transgenic mice. Treatment of ovariectomized females with 17β-estradiol (E2) did not delay the onset of disease, but prevented progression of ALS motor dysfunctions as shown by extension reflex test for a limited time window. Importantly, E2 treatment rescued the lifespans in overiectomized females. These findings will provide important new insights to strategy of ALS treatment.-
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