초록 ▼

연구목표 : 줄기세포의 분자생물학적 기전 연구를 통한 이식줄기세포 활성화 및 제어를 통한 파킨슨병 세포 치료의 임상적용 가능성 제시

연구내용(1단계) :
줄기세포로부터 중뇌도파민신경세포 분화연구
※ 후생학적 유전자조절에 의한 중뇌도파민신경세포 분화유도
※ caRAF를 통한 신경전구 세포 행동조절 및 기전 연구
※ 도파민신경세포 조절인자의 발현시간 조절을 통한 성숙한 도파민신경세포 유도
※ 신경줄기세포 배양 및 도파민신경세포분화 배양에 있어 Insulin이 미치는 영향에 대한 연구
※ Ng

연구목표 : 줄기세포의 분자생물학적 기전 연구를 통한 이식줄기세포 활성화 및 제어를 통한 파킨슨병 세포 치료의 임상적용 가능성 제시

연구내용(1단계) :
줄기세포로부터 중뇌도파민신경세포 분화연구
※ 후생학적 유전자조절에 의한 중뇌도파민신경세포 분화유도
※ caRAF를 통한 신경전구 세포 행동조절 및 기전 연구
※ 도파민신경세포 조절인자의 발현시간 조절을 통한 성숙한 도파민신경세포 유도
※ 신경줄기세포 배양 및 도파민신경세포분화 배양에 있어 Insulin이 미치는 영향에 대한 연구
※ Ngn2, NeuroD와 Nurr1을 통한 도파민신경세포 성숙도 증진 및 형질 유지 연구 기전연구를 통해 도출된 정보 및 기존에 알려진 정보를 사람줄기세포에 적용연구
※ 역분화유도방법 차이에 따른 만능줄기세포 도파민세포 분화비교 연구
※ 중뇌도파민신경세포분화, 생존인자 발현유도에 의한 최적 사람 도파민신경세포 분화 및 이식연구

연구내용(2단계) :
1. 기전연구를 통해 도출된 정보 및 기존에 알려진 정보를 사람줄기세포에 적용연구
※ 사람배아줄기세포 및 사람유도만능줄기세포에서 Doxycycline에 의한 생존 및 증식에 관한 연구
2. 줄기세포분화, 생존, 이식 후 종양형성, 분화된 도파민신경세포 성숙 및 형질유지에 관한 기전연구
- 줄기세포로부터 중뇌도파민신경세포 분화연구
※ Ngn2, NeuroD와 Nurr1을 통한 도파민신경세포 성숙도 증진 및 형질 유지 연구
※ Gene manipulation을 통한 astrocyte의 neural stem cell 생존증진에 관한 연구
※ 유전자조작을 통한 파킨슨모델 생체 내 도파민 신경세포의 생존율 증가 유도에 관한 연구
※ 신경줄기세포에서 발현벡터와 중뇌발달을 고려한 유전자조작으로 기능적인 중뇌도파민신경세포로의 분화
※ 섬유아세포로부터 기능성 도파민신경줄기세포 직접분화

(출처 : 보고서 요약서 3p)

Abstract ▼

IV. Results
(Part1)
1. Study for efficient generation of induced pluripotent stem cells(iPSCs)-derived dopamine neuron
- iPSCs, we use in our exp, are differentiated to DA neuron.
- Viability of protein-based iPSCs, among 7-8 types of iPSCs, was higher than another types of iPSCs, senesc

IV. Results
(Part1)
1. Study for efficient generation of induced pluripotent stem cells(iPSCs)-derived dopamine neuron
- iPSCs, we use in our exp, are differentiated to DA neuron.
- Viability of protein-based iPSCs, among 7-8 types of iPSCs, was higher than another types of iPSCs, senescence was not shown in protein-based iPSCs, but another iPSCs were finally undergone apoptosis for doing several passages.
- In hemi-parkinson rat, rotational behavior tests were improved in rat-transplanted protein-based iPSCs comparing non-treated rat.
2. Mechanism study for Nurr1􌝆Foxa2 collaborative roles in midbrain development
- Foxa2 is required for efficient induction of DA neuron as a epigenetic regulator interacting with Nurr1
- CoREST, repressor of neuronal differentiation, is repelled from Nurr1 interaction by Foxa2 induction in neural precursors, finally enhanced A9 type DA neuronal differentiation, especially TH and DAT promoters.
3. Mechanism study for dopaminergic neuron differentiation by KCl depolarization in ventral midbrain neural precursors(VM-NPCs)
- Mature DA neuronal differentiation is enhanced in VM-NPCs by KCl depolarization
- DA neuronal differentiation is caused by the changes of epigenetic regulation, CoREST, HAT etc.
4. Study for enhanced neurogenic potential of neural precursors by secreted molecules from caRAF transduced neural precursors
- caRAF overexpression in neural precursors were shown two different regulation of neural lineages, neuronal differentiation and glial differentiation(radial glia), respectably, intrinsically and extrinsically.
- Interestingly, caRAF conditioned media(extrinsically) induced radial glia-like cells, which has neurogenic potential.
5. Study for induction of mature DA neuron by decreasing and delaying exogenous Nurr1 expression
- Control for expression level and time of Nurr1 is one of critical factor for generating A9 type DA neuron
- Successed to mimic in vitro DA neuronal differenation culture as a midbrain development using Doxycycline inducible Nurr1 expression vector
6. Mechanism study for the interplays of Ngn2, NeuroD and Nurr1 in midbrain development
- Successed to mimic in vitro DA neuronal differentiation culture as a midbrain development for delaying Nurr1 expression than Ngn2 expression
- Time line of Ngn2/Nurr1 expression in midbrain is important to induce a mature DA neuron

(Part2)
1. Study for proliferation and survival of human embryo stem cells(hESCs) and iPSCs by Doxcycline
- Difined increased proliferation and survival of hESCs and iPSCs by Doxycycline
- Prove for increasing pluripotency gene and stable stem cell characteristic
2. Mechanism study for the interplays of Ngn2, NeuroD and Nurr1 in midbrain development
- Time line of expression of Nurr1, Ngn2 and NeuroD1 approaching to developmental and locational part
- Interaction of transcription and translation step in dopaminergic neuron by siRNA Neurogenin2, NeuroD1
3. Study for enhanced survival of neural stem cells of astrocyte using gene manipulation
- Develop various promoter for lenti virus
- Established method of neural stem cell co-culture with astrocytes
- Changing of expreesion levels of transcriptional factor by Nurr1/Foxa2
4. Study for increased survival of dopaminergic neurons in PD model using gene manipulation
- Enhanced survival dopaminergic neuron for inhibition of apoptotic pathway of dopaminergic neuron by Nurr1/Foxa2
5. Study for differentiation to functional dopaminergic neuron using stable expression vector of neural stem cell and gene manipulation considering developmental system
- Defined lenti/ retro viral system for adapted our gene expression pattern
- Set-up Nurr1 and Foxa2 expression condition as same as in vivo development condition
6. Method of direct differentiation to functional dopaminergic neuron from fibroblast
- Find candidate for related cell proliferation factor in neurogenesis
- We detected Nestin-GFP levels as made various candidate factor retro virus, BCLxL, AKT1, Raf, Wnt1, Dlx2, Otx2
- When BCLxL retro virus express with BAM, cell proliferation increased compare only BAM.
- Co-labeled TH positive neuron with neuron maker as TuJ1, MAP2, HuCD, related dopamine synthesis factors, DAT, VMAT2, and midbrain specific marker as Foxa2, Pitx3, Nurr1. Finally A9 type midbrain marker, Girk2 too.

(출처 : SUMMARY 8~10p)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 4SUMMARY ... 7CONTENTS ... 11목차 ... 11제1장 연구개발과제의 개요 ... 12제2장 국내외 기술개발 현황 ... 15제3장 연구개발수행 내용 및 결과 ... 16제5장 연구개발결과의 활용계획 ... 36제6장 연구개발과정에서 수집한 해외과학기술정보 ... 37제7장 연구시설·장비 현황 ... 38제8장 참고문헌 ... 38끝페이지 ... 40