척수재생을 위한 신경줄기세포와 고분자 지지체를 이용한 복합적인 치료전략의 개발
Development of Combinatorial Strategies Employing Neural Stem Cells and Polymer Scaffold for Spinal Cord Repair
- 주제(키워드) spinal cord injury , neural stem cell , neurotrophin-3 , poly (lactic-co-glycolic acid) scaffolds , serotonergic axon
- 발행기관 아주대학교
- 지도교수 김 병곤
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000009802
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
ABSTRACT- Development of Combinatorial Strategies Employing Neural Stem Cells and Polymer Scaffold for Spinal Cord Repair Stem cell-based therapy holds promise to enhance functional recovery following spinal cord injury (SCI). Most evidence that transplantation of neural stem cells can produce beneficial outcomes after SCI has been derived from rodent models. Before being translated to human patients, it would be needed to examine the therapeutic effects in larger animal models where weight-bearing locomotion should be much more challenging. The present study examined therapeutic effects of a combinatorial strategy centering on human neural stem cells (hNSCs) in a canine hemisection SCI model. After traumatic injuries to the spinal cord, secondary injuries aggravate the extent of damage, expanding cystic cavities at the lesion site. Cavity wall is surrounded by glial scar and severely impedes axonal regrowth. Therefore, any regenerative strategy could be complemented with an attempt to bridge lesion cavities. In the current study, we implanted poly (lactic-co-glycolic acid) (PLGA) scaffolds to facilitate the delivery of neural stem cells. We sought to enhance therapeutic efficacy by ectopic expression of neurotrophin-3 (NT-3) in the hNSCs. NT-3 overexpressing hNSCs (NT3.F3) were produced by retroviral transduction of the immortalized hNSC line (F3) with human NT-3 cDNA. NT3.F3 cells were seeded into a predesigned PLGA (65:35) scaffolds, and the PLGA scaffolds with hNSCs (PLGA-NT3.F3) were implanted immediately after left hemisection at T11 in female dogs weighing 20-30 kg. The PLGA scaffold seemed to nicely fill the lesion cavity, showing a varying degree of biodegradation by 12 weeks. Survival of grafted cells was confirmed at 2 weeks, and some of the grafted cells migrated to the host tissue. There were very few regenerating axons into the scaffold in both groups. Moreover, the ventral horns caudal to the hemisected region were more profusely innervated by serotonergic axons in animals with PLGA-NT3.F3. These findings raise a possibility that implantation of PLGA-NT3.F3 can produce beneficial motor outcomes by promoting axonal remodeling below the lesion. This study suggests that the therapeutic strategy combining multidisciplinary approaches can be feasible and effective for spinal cord repair in larger species. Key words: spinal cord injury; neural stem cells; NT3; PLGA scaffold; serotonergic axon
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES v LIST OF ABBREVIATION vi
I. INTRODUCTION 1
II. MATERIALS AND METHODS 5
1. Culture of immortalized human neural stem cells (hNSCs) 5
2. Pretreatment of scaffold and cell seeding 5
3. ELISA of NT-3 6
4. Spinal cord injury and implantation of scaffold 7
5. Histological preparation . 8
6. Immuohistochemistory 8
7. Quantitative morphometry9
III. RESULTS12
1. Seeding NT-3 overexpressing hNSCs in PLGA scaffold 12
2. Implantation of PLGA seeded with hNSCs (PLGA-NT3.F3) 15
3. Detection of human neural stem cell in scaffold 18
4.Deposition of chondroitin sulfate proteoglycans (CSPG) in the lesion 22
5. Remodeling of serotonergic axons 24
IV. DISCUSSION 29
V. CONCLUSION 33
REFERENCES 34
국문요약 43
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LIST OF FIGURES
Fig. 1. NT-3 overexpressing human neural stem cells 13
Fig. 2. Seeding human neural stem cells (hNSCs) in PLGA Scaffold 14
Fig. 3. Implantation of PLGA seeded with hNSCs (PLGA-NT3.F3) 16
Fig. 4. Area quantifications of spared tissue and residual scaffold 17
Fig. 5. Detection of transplanted human neural stem cells (hNSCs) 19
Fig. 6. Detection of transplanted human neural stem cells (hNSCs) in white matter 20
Fig. 7. Differentiation of neural stem cells in white matter 21
Fig. 8. Deposition of chondroitin sulfate proteoglycans (CSPGs) at the interface 23
Fig. 9. Detection of serotonergic neuron in lesion site by 5-HT staining 26
Fig. 10. Quantification of serotonergic neurons in caudal area : 5-HT staining 27
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LIST OF ABBREVIATION
APC-CC1, anti-APC (Ab-7) mouse mAb (CC-1)
BDNF, brain-derived neurotrophic factor
CNS, central nervous system
CS-56, anti-chondroitin sulfate
CSPG, chondroitin sulfate proteoglycans
DAPI, 4’, 6 –Diamidino-2-Phenylindole Dihydrochloride
DMEM, dulbecco’s modified eagle’s medium
DW, distilledwater
ELISA, enzyme-linked immunosorbent assay
EM, electron microscope
F3, human brain F3
GFAP, anti-glial fibrillary acidic protein
hNSC, human neural stem cell
5-HT, 5-hydroxytryptamine
Hu-Mito, anti-human mitochondria
L, left
NGF, nerve growth factor
NSAID, nonsteroidal antiinflammatory drug
NT3, neurotrophin-3
NT3.F3, neurotrophin-3 overexpressing F3
MAP2, anti-microtubule-associated protein 2
PBS, phosphate buffered saline
PLA, poly lactic acid
PLGA, poly (lactic-co-glycolic acid)
PGA, poly (glycolic acid)
P/S, penicillin streptomycin
R, right
ROI, regions of interest
RT, room temperature
S or SC, scaffold
SCI, spinal cord injury
T, tissue
VH, ventral horn
WM, white matter
