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Effect of brain derived neurotrophic factor-expressing mesenchymal stromal cells and chondroitinaseABC on chronic canine spinal cord injury : 개의 만성 척수 손상에 대한 뇌유래신경인자발현 중간엽줄기세포와 콘드로이치네이즈ABC의 신경 재생 효과

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dc.contributor.advisor권오경-
dc.contributor.author이승훈-
dc.date.accessioned2017-07-13T16:45:50Z-
dc.date.available2017-07-13T16:45:50Z-
dc.date.issued2017-02-
dc.identifier.other000000140684-
dc.identifier.urihttps://hdl.handle.net/10371/120251-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2017. 2. 권오경.-
dc.description.abstractSuccessful repair of spinal cord injury (SCI) is a major issue in veterinary neurosurgery. Multipotent mesenchymal stromal cells (MSCs) have effective potentials of neuronal regeneration. Besides, chondroitinaseABC (chABC) and neuronal factors such as brain-derived neurotrophic factor (BDNF) are widely investigated to repair chronic SCI. Single treatments with those factors, for neurotropic effects or lysis of chondroitin sulfate proteoglycans as barrier to neuronal regeneration have been evaluated. It was hypothesized that combination therapy with factors having different traits could be more effective than single treatment.
The studies were composed of two parts. First, combinational therapy of canine adipose tissue derived MSCs (cADMSCs) and chABC was evaluated in relation to functional recovery and neuronal regeneration. Second, local injections of chABC and BDNF-expressed cADMSCs with intravenous injection of cADMSCs were evaluated.
In the first chapter, it was clarified that 5 U/mL chABC did not have a harmful effect on the viability of cADMSCs. The dogs treated with cADMSCs + chABC and cADMSCs showed significantly better functional recovery 8 weeks after transplantation compared with the negative control and chABC groups (p < 0.05). In addition, the combination of cADMSCs and chABC increased the expression of digested chondroitin sulfate proteoglycans (CSPGs), β3-tubulin, and neurofilament microtuble (NF-M). However, the levels of cyclooxygenase2 (COX2) (p < 0.05) and tissue necrosis factor α (TNFα) were higher in the treatment groups than in the control. Transplantation of cADMSCs + chABC was more effective in improving clinical signs and neural regeneration, but a strategy for anti-inflammation after the treatment for chronic SCI would be needed for further improvement. Higher expressions of inflammation markers could have negative effect on the microenvironment of the injured spinal cord. Neurotrophic factors were not detected even after transplantation of cADMSCs. To improve the clinical outcome after the SCI, the suitable methods that decrease inflammation and increase neurogenic factors are needed.
In the second chapter, combinatorial transplantation of chABC and cADMSCs genetically modified to secrete BDNF with intravenous administration of cADMSCs were investigated. BDNF-expressing MSCs (MSC-BDNF) were generated using a lentivirus packaging protocol. The dogs in the chABC/MSC-BDNF included groups had significantly improved functional recovery 8 weeks after transplantation compared to those in the chABC/MSC-GFP group. The animals in the chABC/MSC-BDNF/IV group showed significant improvements in functional recovery at 6, 7, and 8 weeks compared to those in the chABC/MSC-BDNF group (p < 0.05). Fibrotic changes were significantly decreased in the chABC/MSC-BDNF/IV group. Significant decreases in the expression levels of TNFα, interleukin-6 (IL-6), COX2, gial fibrillary acidic protein (GFAP), and galactosylceramidase (GalC) and increased expression levels of BDNF, β3-tubulin, NF-M, and nestin in the chABC/MSC-BDNF/IV group were observed. These findings suggested that degradation of the glial scar by chABC combined with the presence of neurotrophic factors released by of the transplanted MSCs secreting BDNF can enhance functional recovery after chronic SCI.
It was demonstrated that lentiviral-mediated BDNF gene modification of cADMSCs allowed for stable BDNF production. In addition, chABC combined with cADMSCs transplantation, along with the IV administration of cADMSCs promoted clinical recovery in the injured spinal cord via microenvironment modification, anti-inflammation, and neuronal regeneration. Thus the combinatorial treatment of direct and intraveonus injections of BDNF expressing cADMSCs and chABC has a good therapeutic potential in the treatment of chronic SCI and this can be used as an alternative treatment modality in neuronal regeneration.
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dc.description.tableofcontentsBACKGROUNDS 1
CHAPTER I Effect of the combination of mesenchymal stromal cells and chondroitinaseABC on chronic spinal cord injury 13
ABSTRACT 13
INTRODUCTION 15
MATERIALS AND METHODS 17
1. Isolation and culture of cADMSCs 17
2. Viability of cADMSCs in the presence of chABC 18
3. Duration of chABC activity on CSPGs 18
4. Induction of SCI 19
5. Transplantation of cADMSCs and chABC into the injured sites 20
6. Behavioral assessments 21
7. Histopathological and immunofluorescence analyses 21
8. Western blot analysis 26
9. Statistical analysis 27
RESULTS 28
1. Effect of chABC on cell viability 28
2. Degradation effect of chABC over time 28
3. Behavioral observations 30
4. Histopathological and immunohistochemical assessments 34
DISCUSSION 42

CHAPTER II Impact of local injection of BDNF-expressing mesenchymal stromal cells combined with intravenous delivery mesenchymal stromal cells in canine model of chronic spinal cord injury 47
ABSTRACT 47
INTRODUCTION 49
MATERIALS AND METHODS 51
1. Isolation and culture of cADMSCs 51
2. Lentiviral packing and transfection with green fluorescence protein (GFP) labeled BDNF 52
3. Viability of gene-modified cADMSCs and the effect of chABC 54
4. Western blot analysis of cADMSCs 55
5. Flow cytometry analysis 56
6. Induction of SCI 57
7. Direct transplantation of cADMSCs and chABC into the injured sites and intravenous administration of cADMSCs 58
8. Behavioral assessments 59
9. Histopathological and immunofluorescence analyses 60
10. Western blot analysis 61
11. Statistical analysis 63
RESULTS 64
1. GFP expression and viability of BDNF-expressing cADMSCs 64
2. Effect of chABC on cell viability 64
3. Western blot analysis of the cADMSCs 67
4. Characteristics of gene-modified cADMSCs 67
5. Behavioral observations 71
6. Histopathologic assessments 72
7. Immunohistochemical assessments 78
DISCUSSION 84

GENERAL CONCLUSION 89
REFERENCES 91
국문초록 97
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dc.formatapplication/pdf-
dc.format.extent2166108 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectmesenchymal stromal cells-
dc.subjectcanine spinal cord injury-
dc.subjectchondroitinase ABC-
dc.subjectbrain-derived neurotrophic factor-
dc.subjectintravenous injection-
dc.subject.ddc636-
dc.titleEffect of brain derived neurotrophic factor-expressing mesenchymal stromal cells and chondroitinaseABC on chronic canine spinal cord injury-
dc.title.alternative개의 만성 척수 손상에 대한 뇌유래신경인자발현 중간엽줄기세포와 콘드로이치네이즈ABC의 신경 재생 효과-
dc.typeThesis-
dc.contributor.AlternativeAuthorLee Seung Hoon-
dc.description.degreeDoctor-
dc.citation.pages100-
dc.contributor.affiliation수의과대학 수의학과-
dc.date.awarded2017-02-
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