[보고서]신경계 질환 치료를 목적으로 하는 제대혈 및 지방줄기세포의 다양한 기능 및 역할의 심층 분석

원무호

신경계 질환 치료를 목적으로 하는 제대혈 및 지방줄기세포의 다양한 기능 및 역할의 심층 분석 원문보기

보고서 정보
주관연구기관	강원대학교 Kangwon National University
연구책임자	원무호
보고서유형	2단계보고서
발행국가	대한민국
언어	한국어
발행년월	2012-05
과제시작연도	2012
주관부처	교육과학기술부 Ministry of Education and Science Technology(MEST)
등록번호	TRKO201700011856
과제고유번호	1345176924
사업명	바이오·의료기술개발
DB 구축일자	2017-11-04
키워드	중간엽줄기세포.신경세포분화.내인성 줄기세포.분화조절 인자.치료 효과.Mesenchymal stem cells.neuronal differentiation.endogenous stem cells.trophic factors.therapeutic effects.

초록 ▼

젓간엽 줄기세포의 배양기술을 정립하고 젓간엽줄기세포의 특정 조직세포로의 안정적인 분화유도 방법과 체외 대량으로 줄기세포 증식기법을 활용하여, 제대혈 줄기세포로부터 성공적으로 기능성 신경세포로 분화시켰음. 이는 향후 다른 줄기세포의 신경분화 기법을 개발하는데 있어 젓요한 기초자료로서 활용할 수 있음. 더불어 줄기세포 이식후 이식 줄기세포의 분포와 생존, 신경 및 아교세포로의 분화도를 확인하였으며, 이식 줄기세포가 내인성 신경줄기세포 및 아교세포들의 변화에 영향을 미치는 것에 대한 기초 자료를 확보하였음. 또한 내인성 신경줄기세포를 조절하는 다양한 물질과 기전을 확립하였으며, 최종적으로 줄기세포를 이용하여 신경질환 모델 젓, 뇌허혈, 척수손상 및 좌골신경손상 모델동물들에 적용하여 신경세포 보호 효과 및 치료 효과를 확인함으로써 향 후 줄기세포의 이식 효능에 대한 연구에 있어 기초자료로 활용될 것으로 기대하며, 마지막으로 내인성 신경줄기세포의 증식과 분화 조절하는 인자들에 대한 결과물은 향후 줄기세포제와 병행치료 기법 연구에 있어 매우 젓요한 자료로서 활용될 것으로 기대됨.
(출처: 보고서 요약서 3p)

Abstract ▼

4. Results
가. Establishment of effective methods for mesenchymal stem cells differentiation into neurons in some in vitro systems
- We have established the effective methods for mesenchymal stem cells differentiation into neural cells in an in vivo condition.
- We have founded various factors for the effective differentiation of mesenchymal stem cells into neurons in an in vitro cell culture.
- We have identified the real function of differentiated motor neurons from mesenchymal stem cells

나. Identification of transplanted mesenchymal stem cells distribution and differentiation into neurons and glial cells in some in vivo models
- We have identified a distribution of transplanted mesenchymal stem cells in the brain and spinal cord of the normal mouse, adult and aging gerbils and rats.
- We have identified their survival and differentiation into neurons in the brain and spinal cord of normal mouse, adult and aging gerbils and rats via various routes.
- We have identified the differentiation of transplanted mesenchymal stem cells into glial cells in the brain and spinal cord.

다. Identification of therapeutic effects of transplanted stem cells in some animal models of some CNS diseases
- We have established therapeutic effects of mesenchymal stem cells, neural stem cells on recovery from spinal cord injury, sciatic nerve injury, brain ischemia and aged brain models.
- We have identified the distribution and survival of transplanted mesenchymal stem cells in injured brain and spinal cord in the mouse, adult and aging gerbils and rats.
- We have identified their survival and neuronal differentiation into neurons in injured brain and spinal cord of adult and aging gerbils and rats.
- We have identified the differentiation of mesenchymal stem cells into glial cells in injured brain and spinal cord of adult and aging gerbis and rats.

라. Identification of interaction between transplanted stem cells, endogenous neural stem cells and glial cells
- We have identified effects of transplanted mesenchymal stem cells on endogenous stem cells proliferation and differentiation into neuroblasts and neurons.
- We have identified effects of transplanted mesenchymal stem cells via various routes on glial cell changes in the normal and injured brains.

마. Identification of useful target and methods
- We have found various factors (hormones, drugs, genes) for the regulation of endogenous neural stem cells proliferation and differentiation into neurons.
- We have established some methods for the transplantation of mesenchymal cells in the brain and spinal cord.
- We have identified several mechanisms of the regulation of various factors of endogenous neural stem cells proliferation and differentiation into neurons.
(출처: Summary 10p)

목차 Contents

표지 ... 1제 출 문 ... 2요 약 문 ... 4SUMMARY ... 8CONTENTS ... 12목차 ... 13제1장 연구개발과제의 개요 ... 14제2장 국내외 기술개발 현황 ... 18제3장 연구개발수행 내용 및 결과 ... 20 1절 연구개발 수행 내용 ... 20 2절 연구개발 수행 결과 ... 25제4장 연구개발결과의 활용계획 ... 84제5장 연구개발과정에서 수집한 해외과학기술정보 ... 85제6장 연구시설·장비 현황 ... 87제7장 참고문헌 ... 88끝페이지 ... 88

표/그림 (88)

표 제대혈 유래 줄기세포에서 신경세포 분화 유도.
표 제대혈로부터 motor neuron 분화 프로토콜
표 Motor neurons 분화 유도 후 면역형광염색을 통하여 발현한 신경세포분화 인자 조사.
표 신경분화 조절인자 처치 시 신경세포의 발현 양상 확인.
표 제대혈 유래 줄기세포 배양액을 신경줄기세포 유래 분화세포에 처치 시 내향성전류가 관류함.
표 중간엽유래 줄기세포에서 신경세포 분화 유도 후 specific marker 발현.
표 제대혈 유래 줄기세포에서 신경세포 관련인자 발현.
표 제대혈 유래 줄기세포를 신경분화배지를 이용하여 분화시 neurotrophic factor의 발현조사.
표 Retinoic acid에 의한 신경세포 관련인자의 발현 증가.
표 Shh에 의한 신경세포 관련인자 발현 증가.
표 Retionic acid와 Shh를 동시에 처치하였을 때 신경세포 관련인자 발현 증가.
표 Western blot을 이용한 Sox2와 신경세포 관련인자의 발현양상 분석.
표 제대혈 유래 줄기세포에서 신경세포 분화유도시 Sox2 관련 기전 조사.
표 정상 마우스에 제대혈 유래 줄기세포 이식시 Purkinje 세포와 신경전구체로 marker 발현.
표 제대혈 유래 줄기세포를 이식하였을 때 PKH26의 발현 조사.
표 정상마우스 제대혈 유래 줄기세포의 in vivo survival rate.
표 Fluorescence image of in vitro labeled of Ad-MSC swith CM-DiI. Scale Bar = 25 μm(A-C).
표 Immunofluorescence detection of CM-DiI labeled Ad-MSCs in the hippocampus (A-C), diencephalon (D-E), midbrain (G-H) and spinal cord (J-L).
표 Fluorescence detection of CM-DiI labeled Ad-MSC in the cerebellum (A-H) and the medulla oblongata (I-P) 23 days after Ad-MSC transplantation. Low magnification photos of the engrafted CM-DiI labeled Ad-MSC (red, A and I), the cerebellum and medulla oblongata (bright field, B and J).
표 Cresyl violet staining. Arrows indicates region of transplanted MSCs location.
표 젓간엽줄기세포 실질 이식후 이식젓간엽 실질내 줄기세포의 분포 (Confocal analysis).
표 Double-immunohistofluorescent analysis for GFP and NeuN 14 days after transplantation in the hippocampal CA1. Arrows indicate GFP labeling MSCs.
표 실질 내 중간엽줄기세포 이식 후 아교세포 분화 확인.
표 Cresyl violet staining in the hippocampus 7 days (17 post injury) after MSCs transplantation. Right 2 columns shows high magnification photos of A and C respectively.
표 Fluorescent Anlaysis of GFP tagging MSCS in the ischemic injured region (CA1) and dentate gyrus(DG) of the aging ischemic brain.
표 Differentiation of transplanted MSCs into microglia in the injured region.
표 Differentiation of transplanted MSCs into astrocyte in the dendate gyrus(DG) of the aging ischemic model.
표 Double-immunohistofluorescent analysis for GFP and Doublecortin (DCX) 14 days after transplantation in the hippocampal CA1.
표 Double-immunohistofluorescent analysis for GFP and NeuN 14 days after transplantation in the hippocampal CA1.
표 Double-immunohistofluorescent analysis for GFP and GFAP 14 days after transplantation in the hippocampal CA1.
표 Double-immunohistofluorescent analysis for GFP and Iba-1 14 days after transplantation in the hippocampal CA1.
표 Differentiation of transplanted MSCs into microglia in the injured region.
표 Behavioral testing and functional recovery by grafted hNSC after SCI.
표 Differentiation of transplanted F3.NSC in the injured region of the rat spinal cord 35 days after the transplantation.
표 Immunohistochemical staining for GAP-43 in the SCI-sham (A-C) and SCI-ST (D-L) groups 35 days after the transplantation.
표 Confocal analysis shows that PRVs (A and D) are localized in both the HM positive cells (arrows, C) and NF positive cells (arrows, F) in the injured site of the rat spinal cord.
표 The quantity of labeled cells were counted in each group of nuclei and presented as: +, a few, 1 to 20; ++, moderate, 20 to 100; +++, abundant, 100 to 300; ++++, extremely abundant, more than 300 labeled neurons per section.
표 Distribution of PRV-Ba labeled neurons in the brain after the sciatic nerve injection.
표 Gross anatomy of sciatic nerve in the normal and injured sciatic nerves.
표 Histological analysis normal(A and B) and injured sciatic nerves(C and D) in the rat.
표 Histological analysis injured sciatic nerves 7 days after injury in the rat.
표 Immunohistochemical analysis of myelin basic protein (MBP) 7 days after sciatic nerve injury in the rat.
표 Movement of hind limb in the control-group.
표 Movement of hind limb 1 d days after sciatic nerve inury in the vehicle-(SNI-Sham) and MSCs(SNI-ST) -treated group.
표 Movement of hind limb 30 days after sciatic nerve inury in the vehicle treated group.
표 Necrotic damage (arrows) in hind foot 30 days after sciatic nerve injury in the vehicle-treated group.
표 Movement of hind limb 30 days after sciatic nerve inury in the MSCs treated-group.
표 Rota rod test before and after sciatic nerve injury.
표 Immunohistochemistry for growth associated-protein 43 (GAP43) in the SNI-sham and SNI groups.
표 Panoramic view of panel G in the 그림 50.
표 중간엽줄기세포 이식 후 해마에서의 내인성 줄기세포 증가.
표 중간엽줄기세포 이식 후 해마에서의 내인성 줄기세포 증가.
표 중간엽줄기세포 이식 후 내인성 신경모세포 발현 증가.
표 젓간엽줄기세포 이식 후 내인성 신경모세포 발현 증가.
표 젓간엽줄기세포 이식 후 신경모세포 관련 단백질 증가.
표 중간엽줄기세포 이식 후 해마에서의 내인성 줄기세포 증가.
표 중간엽 줄기세포 뇌실이식후 신경모세포의 발현 증가.
표 Immunohistofluorescent analysis for doublecortin (DCX) after MSCs transplantation in the hippocampal CA1.
표 젓간엽줄기세포 이식후 해마에서의 미세아교세포 발현변화.
표 젓간엽줄기세포 이식 후 해마에서의 별아교세 포 변화.
표 Immunohitochemistry of Iba-1-microglia after MSC transplantation in the brain.
표 Microphotographs of Ki67 immunoreactivity in the dentate gyrus in vehicle- (A and C) and celecoxib-treated (B and D) groups.
표 Microphotographs of DCX immunoreactivity in the dentate gyrus in vehicle- (A and C) and celecoxib-treated (B and D) groups. There are many DCX immunoreactive neuroblasts (arrows) with processes in the subgranular zone of the dentate gyrus.
표 Representative microphotographs for Ki67 Immunohistochemistry in the DG in the age-matched control (A), GD14.5 (B), GD16.5 (C) and GD18.5 (D) group. Ki67+ cells (arrows) are detected in the DG in the control group.
표 Representative microphotographs for DCX+ neuroblasts in the DG in the age-matched control (A and B), GD14.5 (C and D), GD16.5 (E and F) and GD18.5 (G and H) group.
표 Western blot analysis of DCX in the hippocampi of animals in the age-matched control, GD14.5, GD16.5 and GD18.5 group. The relative optical density (ROD) of immunoblot bands are demonstrated as percent values (n = 5 per group; aP < 0.05, significantly different from the age-matched control group, bP < 0.05, significantly different from the GD14.5 group, cP < 0.05, significantly different from the GD16.5 group).
표 Immunohistochemistry for NeuN and F-J B in the DG of the vehicle- (a, c) and CsA-treated (b, d) groups. GCL granule cell layer; ML molecular layer; PL polymorphic layer.
표 Immunohistochemistry for Ki67 in the DG of the vehicle- (a) and CsA-treated (b) groups. GCL granule cell layer; ML molecular layer; PL polymorphic layer.
표 Immunohistochemistry for DCX in the SZDG of the vehicle- (a, b) and CsA-treated (c, d) groups.
표 Immunohistochemistry for Ki67 in the DG of the vehicle- (a), 50 (b) and 200 mg/kg (c) MOE-groups. In all the groups, Ki67 positive nuclei (arrows) are detected in the DG.
표 Immunohistochemistry for DCX in the DG of the vehicle- (a and b), 50 (c and d) and 200 mg/kg (e and f) MOE-groups.
표 Relative number of DCX-immunoreactive neuroblasts with/without tertiary dendrites in the DG (n = 7 per group; aP<0.05, versus the vehicle-group; bP<0.05, versus the 50 mg/kg MOE group).
표 Double immunofluorescence micrographs for BrdU (green) and CB (red) in the DG of the vehicle- (a􍾢 -c) and 200 mg/kg MOE (d􍾢 -f) groups.
표 Serum corticosterone levels in the vehicle-, 50 and 200 mg/kg MOE-groups (n = 7 per group; aP<0.05, versus vehicle-treatedgroup; bP<0.05, versus 50 mg/kg MOE-treated group).
표 Western blot analyses of GAD67 and GABA-T in the DG of the vehicle-, 50 and 200 mg/kg MOE-groups.
표 Immunohistochemistry for Ki67 in the DG of the vehicle- (a), PEP-1- (b), 100 LG (c), and 500 lg (d) PEP-1- SOD1-treated groups. Ki67-positive cells (arrows) are mainly detected in the SGZ. Ki67-positive cells are similar between the vehicleand PEP-1-treated groups.
표 Immunohistochemistry for DCX in the DG of the vehicle- (a, b), PEP-1- (c, d), 100 lg (e, f), and 500 lg (g, h) PEP-1-SOD1-treated groups. DCX-positive neuroblasts have well-developed dendrites (arrows) in the SGZ in the vehicle-treated group.
표 The number of DCX-immunoreactive neuroblasts with/without tertiary dendrites per section of the vehicle-, PEP-1-, 100, and 500 lg PEP-1-SOD1-treated groups
표 Immunohistochemistry for BrdU (a, c, e) and Ki67 (b, d, f) in the DG of the vehicle- (a, b), 1 mg/kg (c, d) and 5 mg/kg (e, f) Tat-SAG-groups. BrdU and Ki67 positive cells (arrows) are observed in the subgranular zone. BrdU positive cells are more abundant in 5 mg/kg Tat-SAG-groups than the vehicle-group, while Ki67 positive cells are more abundant in the 1 and 5 mg/kg Tat-SAG-groups than the vehicle-group.
표 DCX immunohistochemistry in the DG of the vehicle- (a, b), 1 mg/kg (c, d) and 5 mg/kg (e, f) Tat-SAG-groups.
표 Western blot analysis of DCX in the DG of the vehicle-, and 5 mg/kg Tat-SAT-treated groups.
표 4-HNE immunohistochemistry in the DG of the vehicle- (a, b), 1 mg/kg (c, d) and 5 mg/kg (e, f) Tat-SAG-groups.
표 Immunohistochemistry for 8-OHdG in the DG of the vehicle-(a, b), 1 mg/kg (c, d) and 5 mg/kg (e, f) GCL granule cell layer, ML molecular layer, PL polymorphic layer.
표 Differentiation of transplanted mesenchymal stem cell into double immunoreactive neuroblast 30 days after ischemia-reperfusion in the aging gerbi.
표 Differentiation of transplanted mesenchymal stem cell(GFP-SC) into Neuron
표 Differentiation of transplanted mesenchymal stem cells (GFP-SC) into microglia (iba-1) in the hippocampal CA1 region 30 days after ischemia-reperfusion in the aging gerbil.
표 Differentiation of transplanted mesenchymal stem cell(GFP-SC) into astrocytes (GFAP) in the hippocampal CA1 region 30 days after ischemia-reperfusion in the aging gerbil.
표 Daily evaluation of Gait Functional analysis of sciatic nerve injury model after transplantation of mesenchymal stem cells in the injured region.

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신경계 질환 치료를 목적으로 하는 제대혈 및 지방줄기세포의 다양한 기능 및 역할의 심층 분석 원문보기