臺灣博碩士論文加值系統

中文摘要
目的：神經營養因子是失去神經支配的肌肉，再生時所不可或缺的重要因素。目前研究指出，人類羊水幹細胞（AFS）具有分泌多種神經營養因子的潛力，能夠協助神經的再生。在這項研究中，我們將羊水幹細胞，注射到完全去神經化及神經縫合，這兩種動物模式的肌肉中，藉此來評估神經肌肉的再生。
材料與方法：我們使用體重200∼250克的SD大鼠進行這項研究。在完全去神經化的動物模式中，將坐骨神經進行橫向切除，並將其近心端縫到臀部肌肉上。神經縫合的動物模型，進行橫向切除坐骨神經後將兩端縫合4針。用微量注射器將5×106羊水幹細胞注射到腓腸肌中。
結果：睫狀神經營養因子（CNTF）、腦源性神經滋養因子（BDNF）、神經滋養因子3（NT-3）和神經膠質細胞神經營養因子（GDNF）在羊水幹細胞中顯著的表達。肌肉內注射的羊水幹細胞，顯著表達一些神經營養因子，在神經和受神經支配的肌肉中。羊水幹細胞造成失去神經支配的肌肉，增加B-細胞淋巴瘤基因2（Bcl-2）的表現量，並減少Bcl-2相關死亡促進蛋白（Bad）和Bcl-2相關X蛋白（Bax）的表現量。羊水幹細胞能協助維持肌肉的形態，與之相匹配的標的是結蛋白（Desmin）及乙醯膽鹼受體（acetylcholine receptor）的高表現量。羊水幹細胞注射後，神經功能得到顯著改善如CatWalk步態分析，以及神經傳導速率（Latency） 和複合肌肉動作電位波（compound muscle action potential）。有效的改善前角細胞和增加神經的髓鞘及下游的肌肉形態。
結論：羊水幹細胞注射於肌肉後，分泌多種神經營養因子能夠防止肌肉細胞凋亡。這種保護在長期的神經縫合模式中，可以進一步提高神經與肌肉的再生。

Abstract
Purpose: Neurotrophic factors provide the basis for neurotrophic signaling within muscle compartments essential for muscle regeneration in muscle denervation. It is well known that human amniotic fluid mesenchymal cells (AFS) have the potential to secrete various neurotrophic factors mandatory for nerve regeneration. In this study, we assess the outcome of nerve regeneration by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model.
Materials and methods: Sprague-Dawley rats weighting 200-250 gm were enrolled in this study. Muscle denervation was conducted by transverse resection of sciatic nerve with proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of sciatic nerve followed by 4 stitches suture. AFS with 5x106 cells were intramuscularly injected to gastrocnemius muscle with infusion pump.
Results: CNTF, BDNF, NT-3 and GDNF were remarkably expressed in AFS cells. Intra-muscular injection of AFS exerted significantly expression of several neurotrophic factors over nerve and innervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology paralleling with high expression of desmin and acetylocholine receptors. AFS injection produced the significant improvement in neurobehavior such as CatWalk gait analysis as well as nerve conduction latency and CMAP. Significant perseveration of anterior horn cell and increased nerve myelination was line with muscle morphology.
Conclusion: Intramuscular injection of AFS protects muscle apoptosis by the secretion of various neurotrophic factors. This
protection furthermore improves the nerve regeneration in long
term nerve anastmosis model.

目次
中文摘要..............................................................i
英文摘要.............................................................iii
目次.................................................................v
圖表目次............................................................viii
1 序論..............................................................1
1.1 周邊神經損傷後骨骼肌萎縮的機制與再生作用.....................1
1.1.1 骨骼肌萎縮的機制..........................................1
1.1.2 骨骼肌的再生作用..........................................1
1.2 幹細胞的應用.................................................2
1.2.1 幹細胞治療................................................2
1.2.2 羊水幹細胞................................................3
1.3 神經與肌肉再生的評估.........................................3
1.3.1 免疫螢光染色..............................................3
1.3.2 西方點墨法（Western blot）....................................4
1.3.3 電生理....................................................4
1.3.4 CatWalk-步態分析...........................................4
1.3.5 逆行神經示踪..............................................4
1.4 研究動機.....................................................5
2 材料方法..........................................................6
2.1 羊水幹細胞的培養.............................................6
2.2 神經縫合手術.................................................6
2.3 冷凍切片.....................................................8
2.4 免疫螢光染色.................................................8
2.5 西方點墨法...................................................9
2.6 電生理......................................................10
2.7 CatWalk-步態分析.............................................10
2.8 逆行神經示踪................................................10
3 實驗設計.........................................................11
3.1 第一組實驗設計..............................................12
3.2 第二組實驗設計..............................................12
4 實驗結果.........................................................13
4.1 羊水幹細胞在體外及體內蛋白質的表現..........................13
4.2 肌肉完全去神經化的動物模式中肌肉的再生狀況..................14
4.3 神經縫合的動物模式中神經與肌肉的恢復情形....................15
4.4 神經縫合的動物模式中老鼠的運動功能恢復程度..................16
4.5 結論........................................................17
5 討論.............................................................18
6 參考文獻.........................................................22
結果圖表............................................................27

1. H. Jin, Z. Wu, T. Tian, Y. Gu, Apoptosis in atrophic skeletal muscle induced by
brachial plexus injury in rats. The Journal of trauma 50, 31 (Jan, 2001).
2. Z. Z. Gu et al., Gene expression and apoptosis in the spinal cord neurons after
sciatic nerve injury. Neurochemistry international 30, 417 (Apr-May, 1997).
3. L. Sun et al., Cathepsin B-dependent motor neuron death after nerve injury in
the adult mouse. Biochemical and biophysical research communications 399,
391 (Aug 27, 2010).
4. A. Kanamori et al., Superoxide is an associated signal for apoptosis in axonal
injury. Brain : a journal of neurology 133, 2612 (Sep, 2010).
5. P. J. Adhihetty, M. F. O'Leary, B. Chabi, K. L. Wicks, D. A. Hood, Effect of
denervation on mitochondrially mediated apoptosis in skeletal muscle. Journal
of applied physiology 102, 1143 (Mar, 2007).
6. D. L. Allen et al., Apoptosis: a mechanism contributing to remodeling of skeletal
muscle in response to hindlimb unweighting. The American journal of
physiology 273, C579 (Aug, 1997).
7. P. S. Brookes, Y. Yoon, J. L. Robotham, M. W. Anders, S. S. Sheu, Calcium, ATP,
and ROS: a mitochondrial love-hate triangle. American journal of physiology.
Cell physiology 287, C817 (Oct, 2004).
8. M. Crompton, The mitochondrial permeability transition pore and its role in cell
death. The Biochemical journal 341 ( Pt 2), 233 (Jul 15, 1999).
9. N. Joza et al., Essential role of the mitochondrial apoptosis-inducing factor in
programmed cell death. Nature 410, 549 (Mar 29, 2001).
10. P. Li et al., Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade. Cell 91, 479 (Nov 14, 1997).
11. S. A. Susin et al., Molecular characterization of mitochondrial
apoptosis-inducing factor. Nature 397, 441 (Feb 4, 1999).
12. N. J. Waterhouse et al., Cytochrome c maintains mitochondrial transmembrane
potential and ATP generation after outer mitochondrial membrane
permeabilization during the apoptotic process. The Journal of cell biology 153,
319 (Apr 16, 2001).
13. T. W. Sedlak et al., Multiple Bcl-2 family members demonstrate selective
dimerizations with Bax. Proceedings of the National Academy of Sciences of the
United States of America 92, 7834 (Aug 15, 1995).
14. F. C. Ip, J. Cheung, N. Y. Ip, The expression profiles of neurotrophins and their
receptors in rat and chicken tissues during development. Neuroscience letters
23
301, 107 (Mar 30, 2001).
15. E. V. Pitts, S. Potluri, D. M. Hess, R. J. Balice-Gordon, Neurotrophin and
Trk-mediated signaling in the neuromuscular system. International
anesthesiology clinics 44, 21 (Spring, 2006).
16. P. W. Sheard, K. Musaad, M. J. Duxson, Distribution of neurotrophin receptors in
the mouse neuromuscular system. The International journal of developmental
biology 46, 569 (2002).
17. C. Clow, B. J. Jasmin, Brain-derived neurotrophic factor regulates satellite cell
differentiation and skeltal muscle regeneration. Molecular biology of the cell 21,
2182 (Jul 1, 2010).
18. P. Ernfors, K. F. Lee, J. Kucera, R. Jaenisch, Lack of neurotrophin-3 leads to
deficiencies in the peripheral nervous system and loss of limb proprioceptive
afferents. Cell 77, 503 (May 20, 1994).
19. S. Davis et al., Released form of CNTF receptor alpha component as a soluble
mediator of CNTF responses. Science 259, 1736 (Mar 19, 1993).
20. M. D. Grounds, K. E. Davies, The allure of stem cell therapy for muscular
dystrophy. Neuromuscular disorders : NMD 17, 206 (Mar, 2007).
21. B. Peault et al., Stem and progenitor cells in skeletal muscle development,
maintenance, and therapy. Molecular therapy : the journal of the American
Society of Gene Therapy 15, 867 (May, 2007).
22. C. A. Collins et al., Stem cell function, self-renewal, and behavioral
heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289
(Jul 29, 2005).
23. J. Meng, C. F. Adkin, S. W. Xu, F. Muntoni, J. E. Morgan, Contribution of human
muscle-derived cells to skeletal muscle regeneration in dystrophic host mice.
PloS one 6, e17454 (2011).
24. C. De Bari et al., Skeletal muscle repair by adult human mesenchymal stem cells
from synovial membrane. The Journal of cell biology 160, 909 (Mar 17, 2003).
25. M. S. Tsai et al., Clonal amniotic fluid-derived stem cells express characteristics
of both mesenchymal and neural stem cells. Biology of reproduction 74, 545
(Mar, 2006).
26. H. C. Pan et al., Enhanced regeneration in injured sciatic nerve by human
amniotic mesenchymal stem cell. Journal of clinical neuroscience : official
journal of the Neurosurgical Society of Australasia 13, 570 (Jun, 2006).
27. H. C. Pan et al., Post-injury regeneration in rat sciatic nerve facilitated by
neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.
Journal of clinical neuroscience : official journal of the Neurosurgical Society of
24
Australasia 14, 1089 (Nov, 2007).
28. H. C. Pan et al., Combination of G-CSF administration and human amniotic fluid
mesenchymal stem cell transplantation promotes peripheral nerve
regeneration. Neurochemical research 34, 518 (Mar, 2009).
29. H. C. Pan et al., Human amniotic fluid mesenchymal stem cells in combination
with hyperbaric oxygen augment peripheral nerve regeneration. Neurochemical
research 34, 1304 (Jul, 2009).
30. F. C. Cheng et al., Enhancement of regeneration with glia cell line-derived
neurotrophic factor-transduced human amniotic fluid mesenchymal stem cells
after sciatic nerve crush injury. Journal of neurosurgery 112, 868 (Apr, 2010).
31. R. Donato, Intracellular and extracellular roles of S100 proteins. Microscopy
research and technique 60, 540 (Apr 15, 2003).
32. Z. Li et al., Desmin is essential for the tensile strength and integrity of myofibrils
but not for myogenic commitment, differentiation, and fusion of skeletal
muscle. The Journal of cell biology 139, 129 (Oct 6, 1997).
33. H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proceedings of the National Academy of Sciences of the United States of
America 76, 4350 (Sep, 1979).
34. J. Renart, J. Reiser, G. R. Stark, Transfer of proteins from gels to
diazobenzyloxymethyl-paper and detection with antisera: a method for
studying antibody specificity and antigen structure. Proceedings of the National
Academy of Sciences of the United States of America 76, 3116 (Jul, 1979).
35. M. Scanziani, M. Hausser, Electrophysiology in the age of light. Nature 461, 930
(Oct 15, 2009).
36. A. Bozkurt et al., CatWalk gait analysis in assessment of functional recovery
after sciatic nerve injury. Journal of neuroscience methods 173, 91 (Aug 15,
2008).
37. R. Deumens, R. J. Jaken, M. A. Marcus, E. A. Joosten, The CatWalk gait analysis
in assessment of both dynamic and static gait changes after adult rat sciatic
nerve resection. Journal of neuroscience methods 164, 120 (Aug 15, 2007).
38. P. O'Donnell, A. Lavin, L. W. Enquist, A. A. Grace, J. P. Card, Interconnected
parallel circuits between rat nucleus accumbens and thalamus revealed by
retrograde transynaptic transport of pseudorabies virus. The Journal of
neuroscience : the official journal of the Society for Neuroscience 17, 2143 (Mar
15, 1997).
39. A. H. Luo, G. Aston-Jones, Circuit projection from suprachiasmatic nucleus to
25
ventral tegmental area: a novel circadian output pathway. The European journal
of neuroscience 29, 748 (Feb, 2009).
40. H. Schmalbruch, W. S. al-Amood, D. M. Lewis, Morphology of long-term
denervated rat soleus muscle and the effect of chronic electrical stimulation.
The Journal of physiology 441, 233 (Sep, 1991).
41. A. S. Burns, V. S. Boyce, A. Tessler, M. A. Lemay, Fibrillation potentials following
spinal cord injury: improvement with neurotrophins and exercise. Muscle &
nerve 35, 607 (May, 2007).
42. F. Pellicer, A. Lopez-Avila, E. Torres-Lopez, Electric stimulation of the cingulum
bundle precipitates onset of autotomy induced by inflammation in rat.
European journal of pain 3, 287 (Jun, 1999).
43. Z. Seltzer, B. Z. Beilin, R. Ginzburg, Y. Paran, T. Shimko, The role of injury
discharge in the induction of neuropathic pain behavior in rats. Pain 46, 327
(Sep, 1991).
44. Z. Seltzer, S. Cohn, R. Ginzburg, B. Beilin, Modulation of neuropathic pain
behavior in rats by spinal disinhibition and NMDA receptor blockade of injury
discharge. Pain 45, 69 (Apr, 1991).
45. M. M. Daadi et al., Functional engraftment of the medial ganglionic eminence
cells in experimental stroke model. Cell transplantation 18, 815 (2009).
46. W. Wu et al., Transplantation of neural stem cells expressing hypoxia-inducible
factor-1alpha (HIF-1alpha) improves behavioral recovery in a rat stroke model.
Journal of clinical neuroscience : official journal of the Neurosurgical Society of
Australasia 17, 92 (Jan, 2010).
47. L. Zhang, S. Gu, C. Zhao, T. Wen, Combined treatment of neurotrophin-3 gene
and neural stem cells is propitious to functional recovery after spinal cord injury.
Cell transplantation 16, 475 (2007).
48. C. J. Chen et al., Transplantation of bone marrow stromal cells for peripheral
nerve repair. Experimental neurology 204, 443 (Mar, 2007).
49. D. Lu et al., Intravenous administration of human umbilical cord blood reduces
neurological deficit in the rat after traumatic brain injury. Cell transplantation
11, 275 (2002).
50. D. E. Erb, R. J. Mora, R. P. Bunge, Reinnervation of adult rat gastrocnemius
muscle by embryonic motoneurons transplanted into the axotomized tibial
nerve. Experimental neurology 124, 372 (Dec, 1993).
51. C. K. Thomas, D. E. Erb, R. M. Grumbles, R. P. Bunge, Embryonic cord transplants
in peripheral nerve restore skeletal muscle function. Journal of neurophysiology
84, 591 (Jul, 2000).
26
52. J. G. Tidball, Inflammatory processes in muscle injury and repair. American
journal of physiology. Regulatory, integrative and comparative physiology 288,
R345 (Feb, 2005).
53. K. Seidl, C. Erck, A. Buchberger, Evidence for the participation of nerve growth
factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic
program. Journal of cellular physiology 176, 10 (Jul, 1998).
54. M. Rende et al., Regulation of the p75 neurotrophin receptor in a rat myogenic
cell line (L6). The Histochemical journal 31, 589 (Sep, 1999).
55. S. Reddypalli et al., p75NTR-mediated signaling promotes the survival of
myoblasts and influences muscle strength. Journal of cellular physiology 204,
819 (Sep, 2005).
56. E. Castren, Is mood chemistry? Nature reviews. Neuroscience 6, 241 (Mar,
2005).
57. L. de Medinaceli, W. J. Freed, R. J. Wyatt, An index of the functional condition of
rat sciatic nerve based on measurements made from walking tracks.
Experimental neurology 77, 634 (Sep, 1982).