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The Feasibility of Amniotic Fluid Mesenchymal Stem Cells in Treatment of Sciatic Nerve Injury
|關鍵字:||amniotic fluid mesenchymal stem cell;羊水間葉幹細胞;sciatic nerve injury;坐骨神經損傷||出版社:||獸醫學系暨研究所||引用:||1. Frostick SP. The physiology and metabolic consequence of muscle denervation. Int Angio 1995; 14: 278-287. 2. Pollock SP. Nerve regeneration. Curr Opinion Neurol 1995; 8: 354-358. 3. Mirsky R, Jessen KR. Schwann cell development, differentiation and myelination. Curr Opninion Neurobiol 1996; 6:89-96. 4. Pannu R, Barbosa E, Singh AK, Singh I. Attenuation of acute inflammatory response by Atorvastatin after spinal cord injury in rats. J Neurosci Res 2005; 79(3): 340-350. 5. Bryan DI, Miller RA, Costas PD, et al. Immunocytochemistry of skeletal muscle basal laminia grafts in nerve regeneration. Plast Reconstr Surg 1993; 92:927-940. 6. Seckel BP. Enhancement of peripheral nerve regeneration. Muscle & Nerve 1990; 13: 785-800. 7. Ho PR, Coan GM, Cheng ET, et al. 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羊水間葉幹細胞( amniotic fluid mesenchymal stem cell)目前已被分離及鑑定。然而使用羊水間葉幹細胞以治療周邊神經損傷的研究非常少。因此本研究主要是研究使用羊水間葉幹細胞，以治療周邊神經損傷的可行性。研究使用羊水間葉幹細胞的可行性主要分成三部分。第一部分：使用同一種系的細胞移植，把大白鼠的羊水間葉幹細胞移植到大白鼠的坐骨神經上。第二部分：使用人類的羊水間葉幹細胞移植到大白鼠的坐骨神經上。第三部分：使用人類的羊水間葉幹細胞移植到大白鼠的坐骨神經上，並使用顆粒球生長激素(granulocyte-colony stimulating factor )來改善移植的環境。
第一部分：左側坐骨神經使用止血夾來造成神經損傷，把大白鼠的羊水間葉幹細胞移植到大白鼠的坐骨神經上。使用免疫螢光分析及組織染色來檢視神經滋養因子的表現。神經功能的評估包括運動功能、電生理、組織及免疫螢光分析。結果顯示大白鼠的羊水間葉幹細胞表現CD29及CD44，但不表現CD11b及CD45。羊水間葉幹細胞同時表現BDNF (brain-derived neurotrophic factor)、GDNF (glia cell line-derived neurotrophic factor)、CNTF (ciliary neurotrophic factor)、 NGF (nerve growth factor)及NT-3 (neurotrophin-3)的神經滋養因子。大白鼠的羊水間葉幹細胞的移植促進神經的生長，表現於運動功能、神經電生理及神經組織的鞘化程度。此外神經組織於免疫螢光分析及免疫染色，發現羊水間葉幹細胞表現及分泌神經滋養因子。
Amniotic fluid mesenchymal stem cells (AFS) have been successfully isolated and identified. The application of AFS in repair of peripheral nerve injury was scarce. This study was conducted to evaluate the feasibility of AFS in the treatment of peripheral nerve injury. The determination of feasibility consisted of the same species transplantation from rat AFS to rat sciatic nerve injury (Part I), human AFS to rat sciatic nerve gap injury (Part II), and human AFS to rat sciatic nerve injury assisted with the improvement of transplantation milieu by granulocyte colony-stimulating factor (G-CSF) (Part III).
Part I: Fifty Sprague Dawley rats weighing from 250-300 gm were used in this study. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in the fibrin glue and woven Surgicel were delivered to injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemical (IHC) studies were used to detect the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and IHC studies. Positive CD29/44, negative CD11b/45 as well as expression of brain-derived neurotrophic factor (BDNF), glia cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) were demonstrated in rat AFS. The motor function, compound muscle action potential, and nerve conduction latency showed significant improvement in those groups treated with AFS. ELISA measurement in retrieved nerves displayed significant elevation of CNTF and NT-3. The IHC studies demonstrated positive staining of CNTF and NT-3 in transplanted AFS. The histology and IHC studies revealed less fibrosis and higher expression of S-100 and GFAP at the injured nerve.
Part II: Twenty Sprague- Dawley rats weighting 250 to 300 gm were entered into this study. The gap model was constructed by excising 5 mm nerve followed by two 4-0 silk laterally interposed to secure the nerve continuity assisted with 9-0 nylon sutured to the epineuria. One group (n=10) received therapy with AFS embedded into the woven Surgicel and fibrin glue while the control (n=10) only received treatment of woven Surgicel and fibrin glue. Evaluation method included behavioral, electrophysiological, and immunohistochemical studies. In gait analysis, the angle of ankle in treatment and control group was 46.4±15 and 36±8.2, respectively, which revealed the statistical significance (p=0.045). Five of 10 rats (50%) demonstrated partial movement of feet, while none of the control group revealed any trace movement. The ratio of compound muscle action potential (CMAP) in the experimental group was 43±12.5% as compared to 29±8.8% in the control group (p=0.038). The conduction latency also showed the reciprocal trend (p=0.005). The histological examination demonstrated that 70% of the treatment group achieved maximum diameter ratio of the nerve gap (>50%), compared with 0% in the control group. There was no significant difference in direction of fiber and fibrosis reaction between groups.
Part III: Peripheral nerve injury was produced in Sprague-Dawley rats by crushing left sciatic nerve using a vessel clamp. AFS were embedded in fibrin glue and woven Surgicel and delivered to the injured site. G-CSF (50
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