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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수의과대학 수의학과
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서울대학교 대학원
mesenchymal stromal cellscanine spinal cord injurychondroitinase ABCbrain-derived neurotrophic factorintravenous injection
학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2017. 2. 권오경.
Successful 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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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Theses (Ph.D. / Sc.D._수의학과)
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