파킨슨 병 모델에서 중간엽 줄기세포의 Neurogenesis 증강효과를 규명한 최초의 연구로 1) in vitro neural progenitor 모델에서 중간엽 줄기세포의 신경독소에 의한 신경보호효과를 규명하였으며 2) 파킨슨 병 동물모델에서 subventricular zone 및 중뇌 흑질에서 현저하게 저하된 neurogenesis가 중간엽 줄기세포 투여후 8일째 및 30일째 이부위에서 현저하게 증가되는 사실을 규명하였으며 3) 특히 중뇌 흑질에서 중간엽 줄기세포 투여 후 증가된 neurogenesis는 도파민성 신경원으로 분화
파킨슨 병 모델에서 중간엽 줄기세포의 Neurogenesis 증강효과를 규명한 최초의 연구로 1) in vitro neural progenitor 모델에서 중간엽 줄기세포의 신경독소에 의한 신경보호효과를 규명하였으며 2) 파킨슨 병 동물모델에서 subventricular zone 및 중뇌 흑질에서 현저하게 저하된 neurogenesis가 중간엽 줄기세포 투여후 8일째 및 30일째 이부위에서 현저하게 증가되는 사실을 규명하였으며 3) 특히 중뇌 흑질에서 중간엽 줄기세포 투여 후 증가된 neurogenesis는 도파민성 신경원으로 분화된다는 사실을 처음으로 규명함. 이러한 연구결과는 파킨슨 병을 포함한 퇴행성 뇌질환에서 저하된 neurogenesis의 복구 치료전략에 중간엽 줄기세포의 역할을 처음으로 규명한 것으로 향후 퇴행성 뇌질환영역의 세포치료에서 또 하나의 임상적용 토대를 마련함과 동시에 치료 방법이 전무했던 다른 뇌질환을 비롯한 성인 질환의 치료제로서의 그 가능성을 높일 것임.
Abstract▼
1. Background and objectives It is known that neurogenesis in the subventricular zone (SVZ) and olfactory bulb was impaired in a dopamine depletion-induced model of Parkinsons disease (PD) and the brains of individuals with PD. Recent studies have demonstrated that mesenchymal stem cells(MSCs) in
1. Background and objectives It is known that neurogenesis in the subventricular zone (SVZ) and olfactory bulb was impaired in a dopamine depletion-induced model of Parkinsons disease (PD) and the brains of individuals with PD. Recent studies have demonstrated that mesenchymal stem cells(MSCs) increase neurogenesis in the SVZ of ischemic animal models though various cytotrophic factors. In this study, we investigated whether human mesenchymal stem cells (hMSCs) increase neurogenesis in the SVZ and the substantia nigra (SN) of MPTP-induced PD animal models. 2. Methods Study To introduce MPTP-induced animal model of PD, C57BL/6 adult male mice aged 16 weeks were injected MPTP (20 mg/kg, i.p., four times a day at 2 h intervals, each group, n=5). Control mice(n=5) were injected with saline alone, using the same administration method. At 3 days after MPTP injection, hMSCs were injected in to thetailvain(1´106cells/ml).At4 days and 25days after MPTP injection, Brd U(50mg/kg, i.p.) was injected for 5days. Histopathological analyses were compared among three groups of mice (control group, only MPTP group, hMSCs treatment in MPTP group). To in vitro system of neurogenesis, we developed primary culture of neuronal precursor cell, and MPP+ was introduced in this system. Isolation of hMSC Bone marrow aspirates (10mL) were obtained from the iliac crests of human donors. The mononuclear cell layer was isolated by Ficoll-Hypaque, washed in PBS, plated in polystyrene plastic 100mm culture dishes, and cultivated in low-glucose Dulbecco modified Eagles medium (Gibco-BRL, Grand Island, NY), containing 10% fetal bovine serum (Hyclone, Irvine, CA) and 1% penicillin/streptomycin (P/S, Sigma, St. Louis, MO) in a humidified incubator at 37°C under 5%CO2. Non-adherent cells were removed after 24h. When the primary cultures reached 80% confluence , the cells were harvested using 0.25% trypsin and subcultured. At passage6, hMSCs were injected into mice via tail vain. Tissue preparation For immunohisotochemistry, the mice were perfused with saline solution containing 0.5% sodium nitrate and heparin (10 U/mL) and were fixed with 4% paraformaldehyde dissolved in 0.1M PB (~ 50 mL/mouse) at 8days and 30 days after first injection. The brains were removed from the skulls, post-fixed overnight in buffered 4% paraformaldehyde at 4℃ and stored in a 30% sucrose solution for 1 to 2 days at 4℃, until they sank. They were then sectioned to obtain a 30μm coronal sections. The 30$\mu$m coronal sections were stored in tissue stock solution (30% glycerol, 30% ethylene glycol, 30% 3 times distilled water, 10% 0.2M PB) at 4℃ until required. For Western blotting, the mice were euthanized at 30 days after first injection, and the SN and striatum area were rapidly removed from the brains and frozen at –70℃. 3. Results ► hMSCs increased significantly survival of NPCs after introduction of MPP+ ► The number of BrdU-ir cells was significanlty decreaed in the SVZ of MPTP-treated animal model of PD. ► However, the hMSCs injection significantly increased the number of BrdU-ir cells in the SVZ and the midbrain in animal model of PD. ► The BrdU-ir cells were also co-merged with mature neuronal marker as well as TH-ir marker, which may represent direct transdifferentiation of dopaminergic neurons from neurogenetic neurons. ► The degree of neurogenesis was more prominent in early stage of hMSCs injection than in later stage. ► The introduction of levodopa and pramipexol increased significantly BrdU-ir cells in the SVZ of animal model of PD, where the number of BrdU-ir cells was higher significanlty in pramipexo-treated animals than in levodopa-treated animals. Conclusions Our study showed that hMSCs augumented neurogenesis in the SVZ and the midbrain in animal model of PD. in this regard, our data may contribute to the new therapeutic strategy based on neurogenesis by cell therapy
목차 Contents
겉표지 ...1
제출문 ...3
보고서 요약서 ...4
요약문 (한글) ...5
요약문 (영문) ...6
연구 성과 실적 및 향후 계획 ...8
참여연구원 현황표 ...12
1. 연구개발과제의 배경 및 필요성 ...13
가.파킨슨병의 치료전략 ...13
2. 국내외 기술개발 현황 ...14
① 퇴행성 질환에서의 중간엽 줄기세포를 이용한 세포대체요법 - replacement therapy 치료 전략의 임상적.현실적 한계 ...14
② 퇴행성 신경계 질환에서 중간엽 줄기세포의 적용 - 재생의학의 관점에서 disease-modifying 치료전략으로의 전환 필요성 ...15
③ 파킨슨 병에서 중간엽 줄기세포의 효과 - 도파민성 신경원의 세포사 미세환경 극복기전 ...15
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