臺灣博碩士論文加值系統

脊髓肌肉萎縮症(spinal muscular atrophy, SMA)，是以脊髓前角細
胞退化為主要病理變化的一種遺傳性疾病，依臨床症狀的嚴重程度可
分為type I、II、III 三型。SMN (survival motor neuron) gene 位於人類
染色體5q13，包含一個SMN1 copy 及數個SMN2 copies；SMA 病人
之SMN1 gene 均為homozygous mutation。SMA小鼠是以基因剔除Smn
小鼠與轉殖人類SMN2 的小鼠交配後，產生SMN2/Smn-/-基因型的
SMA 小鼠，臨床上與人類SMA 相似亦可分為三型，牠們的肌肉及脊
髓變化與人類SMA 病理變化非常類似，是研究人類SMA 之一個最
佳的動物模式。
細胞凋亡是動物細胞一種生理性細胞死亡，在動物身體內經常活
躍地進行。最近的研究顯示凋亡作用存在於神經退化疾患、腦缺血疾
患、神經肌肉疾患及運動神經元疾病。但至目前為止在SMA 病患的
活體組織中缺乏細胞凋亡的直接有力證據。
為了證實凋亡作用存在SMA中，我們以 type I SMA小鼠為實驗
對象，在小鼠死亡前，將小鼠置於冰上直接解剖採取脊髓，以固定液
固定後做石蠟切片，進行H&E、double labeling with TUNEL and Nissl
染色及電子顯微鏡觀察。經顯微鏡觀察及計數，type I SMA mice脊髓之運動神經元之細胞核有DNA fragmentation 及chromatin
condensation 的現象；而且其於胸椎及腰椎之脊髓運動神經元個數比
age-matched control mice及wild type mice之脊髓運動神經元數目少
( P<0.05 )，而且在type I SMA mice中，脊髓運動神經元進行細胞凋亡
的比例明顯高於age-matched control mice及wild type mice
( P<0.001 )。希望藉由證實細胞凋亡在SMA小鼠確實存在，除能了解
SMA之致病生理及病理機轉外，對於日後治療藥物之開發與藥物療
效之觀察，會有不同之探討途徑與更寬廣的視野。

Childhood spinal muscular atrophy (SMA) is an autosomal recessive
neuromuscular disorder, characterized by degeneration of the anterior
horn cells of the spinal cord and by symmetrical proximal muscle
weakness and atrophy. Three forms of SMA have been recognized, based
on varying clinical severity. The survival motor neuron (SMN) gene is
present in humans in a telomeric copy, SMN1, and several centromeric
copies, SMN2. Homozygous mutation of SMN1 is associated with SMA.
We produced mouse lines deficient for mouse Smn and transgenic
mouse lines that expressed human SMN2. In contrast, transgenic mice
harboring SMN2 in the Smn-/- background showed pathological changes
in the spinal cord and skeletal muscles similar to those of SMA patients.
The variable phenotypes of Smn-/-SMN2 mice reflect those seen in SMA
patients, providing a mouse model for this disease.
To investigate the role of apoptosis in SMA. We sacrife the mice
before dying on ice and collect the spinal cords from type I SMA mice.
The amount of apoptosis was assessed by H&E stain, double labeling
with TUNEL (TdT-mediated dUTP-biotin nick end labeling ) and Nisslstainings, and electron microscopy.
The morphological changes in H&E staining revealed that chromatin
condensation and fragmentation in the motorneuron of type I SMA mice
spinal cord. The percentage of apoptosis was higher in SMA mice than in
age-matched control mice and wild type mice. In contrast, the
motorneuron numbers of spinal cord in thoracic and lumbar of type I
IV
SMA mice were less than two control groups. We hope this novel finding
can provide a new insight to the understanding of the pathogenesis of
SMA and the investigation of new therapies for this devastating illness of
childhood.

中文摘要………………………………………………………………I-II
英文摘要…………………………………………………………….III-IV
縮寫表…………………………………………………………………...V
第一章 緒 論……………………………………………………........1
1-1 脊髓肌肉萎縮症之背景說明……………………………………..1
1- 2 脊髓肌肉萎縮症之動物模式…………………………………….5
1- 3 細胞凋亡的特徵………………………………………………….6
1-4 細胞凋亡與神經退化性疾病…………………………………......8
1- 5 細胞凋亡與神經肌肉疾病……………………………………….9
1- 6 細胞凋亡與脊髓肌肉萎縮症…………………………………...10
1- 7 實驗目的………………………………………………………...11
第二章 材料與方法…………………………………………………..13
2- 1 實驗材料………………………………………………………...13
2-2 基因鑑定………………………………………………………...13
2-2-1 萃取DNA……………………………………………………14
2-2-2 聚合酶連鎖反應…………………………………………….14
2-2-3 瓊膠電泳…………………………………………………….16
2-3 檢體製備…………………………………………………………16
2-4 Hematoxylin and eosin（H&E）染色…………………………….17
2-5 TUNEL染色及Nissl 染色………………………………………18
2-6 電子顯微鏡觀察…………………………………………………21
2-7 統計方法………………………………………………………...21
第三章 結果…………………………………………………………..22
第四章 討論…………………………………………………………..27
圖、表…………………………………………………………………..30
參考文獻………………………………………………………………..46

Bishop A, Yet SF, et al. A key role for heme oxygenase-1 in nitric oxide
resistance in murine motor neurons and glia. Biochem Biophys Res
Commun 2004;325:3–9
Migheli A, Atzori C, Piva R, et al. Lack of apoptosis in mice with ALS.
Nat Med 1999;5:966-67
Baghdiguian S, Martin M, Richard I, et al. Calpain 3 deficiency is
associated with myonuclear apoptosis and profound perturbation of the
IkB/NF-κB pathway in limb-girdle muscular dystrophy type 2A. Nat Med
1999;5:503-11.
Bruce AK, Jacobsen E, Dossing H, et al. Hypoglycaemia in spinal
muscular atrophy. Lancet 1995;346:609-10.
Bruzstowicz LM, Lehner T, Castilla LH, et al. Genetic mapping of
chronic childhood-onset spinal muscular atrophy to chromosome
5q11.2-13.3. Nature 1990;344:540-1.
Bürglen L, Seroz T, Miniou P, et al. The gene encoding p44, a subunit of
the transcription factor TFIIH, is involved in large-scale deletions
associated with Werdnig-Hoffmann disease. Am J Hum Genet 1997;60:
72-9.
Chang HC, Hung WC, Chunag YJ, Jong YJ, et al. Degradation of
survival motor neuron (SMN) protein is mediated via the
ubiquitin/proteasome pathway. Neurochem Int 2004;45:1107-12.
Clarke PGH, et al. Developmental cell death: Morphological diversity
and multiple mechanisms. Anat Embryol 1990;181:195-213.
Clarke PGH. Apoptosis versus necrosis: how valid a dichotomy for
neurons. In: Koliatsos VE, Ratan RR. eds. Cell death and disease of the
nervous system. Totowa. New Jersey: Human Press, 1999, pp.3-28.
Clermont O, Burlet P, Burglen L, et al. Use of genetic and physical
mapping to locate the spinal muscular atrophy locus between two new
highly polymorphic DNA markers. Am J Hum Genet 1994;54:687-94.
Cohen JJ, et al. Apotposis. Immunol Today 1993;14:126-30.
Dubowitz V, et al. Chaos in classification of spinal muscular atrophies.
Neuromusc Disord 1991;1:77-80.
Dubowitz V, et al. Muscle disorders in childhood. 2nd ed. WB Saunders:
Philadelphia 1995;12:323-69.
Frijns CJM, Van Deutekom J, Frants RR, et al. Dominant congenital
benign spinal muscular atrophy. Muscle Nerve 1994;17:192-7.
Garrieli Y, Sherman Y, Ben-SassonS A. Identification of programmed cell
death in situ via specific labeling of nuclear DNA fragmentation. J Cell
Biol 1992;119:493-501.
Gilliam TC, Bruzstowicz LM, Castilla LH, et al. Genetic homogeneity
between acute and chronic forms of spinal muscular atrophy. Nature
1990;345:823-5.
Hetts SW, et al. To die or not to die: an overview of apoptosis and its role
in disease. JAMA 1998;279:300-7.
Schmitt HP, Harle M, Koelfen W, et al. Childhood progressive spinal
muscular atrophy with facioscapulo-humeral predominance, sensory and
autonomic involvement and optic atrophy. Brain Dev 1994;16:386-92.
Hsieh-Li HM, Chang JG, Jong YJ, et al. A mouse model for spinal
muscular atrophy. Nat Genet 2000;24:66-70.
Ikezoe K, Yan C, Momoi T, Imoto C, Minami N, Ariga M, Nihei K,
Nonaka I. A novel congenital myopathy with apoptotic changes. Ann
Neurol 2000;47:531-6.
Iwahashi H, Eguchi Y, Yasuhara N, et al. Synergistic anti-apoptotic
activity between Bcl-2 and SMN implicated in spinal muscular atrophy.
Nature 1997;390:413-7.
Jong YJ, Chagn JG, Wu JR, et al. Large-scale deletions in a Chinese
infant associated with a variant form of Werdnig-Hoffmann disease.
Neurology 1998;51:878-9.
Jong YJ, Chang JG, Lin SP, et al. Analysis of the mRNA transcripts of the
survival motor neuron (SMN) gene in the tissue of an SMA fetus and the peripheral blood mononuclear cells of normals, carriers and SMA patients.
J Neurol Sci 2000;173:147-53.
Kostic V, Jackson-Lewis V, de Bilbao F, Dubois-Dauphin M, Przedborski
S. Bcl-2: prolonging life in a transgenic mouse model of familial
amyotrophic lateral sclerosis. Science 1997;277:559-62.
Lawson SJ, Lowrie MB: The role of apoptosis and excitotoxicity in the
death of spinal motorneurons and interneurons after neonatal nerve injury.
Neuroscience 1998;87: 337-348.
Lewin B: Genes for SMA: multum in Parvo. Cell 1995;80:1-5.
Lefebvre S, Bürglen L, Reboullet S, et al. Identification and
characterization of a spinal muscular atrophy-determining gene. Cell
1995;80:155-65.
Liston P, Roy N, Tamai K, et al. Suppression of apoptosis in mammalian
cells by NAIP and a related family of IAP genes. Nature
1996;379:349-53.
Lin SP, Chang JG, Tsai CH, et al. Molecular analysis of SMN, NAIP and
P44 gene of SMA patients and their family members. Am J Hum Genet
1998;63, A371, 2149.
Lorson, C.L., Androphy, E.J, et al.. An exonic enhancer is required for
inclusion of an essential exon in the SMA-determining gene SMN. Hum
Mol Genet 2000;9:259-65.
MacKenzie A, Besner A, Roy N, et al. Rapid diagnosis of infantile spinal
muscular atrophy by direct amplication of amniocyte and CVS DNA. J
Med Genet 1993;30:162-3.
Melki J, Burlet P, Clermont O, et al. Refined linkage map of chromosome
5 in the region of the spinal muscular atrophy gene. Genomics
1993;15:521-4.
Munsat TL: Workshop report-International SMA collaboration.
Neuromus Disord 1991;1:81.
Novelli G, Capon F, Tamisari L, et al. Neonatal spinal muscular atrophy
with diaphragmatic paralysis is unlinked to 5q11.2-q13. J Med Genet
1995;32:216-9.
Olive M, Martinez-Matos JA, Montero J, Ferrer I. Apotposis is not
mechanism of cell death of muscle fibers in human muscular dystrophies
and inflammatory myopathies. Muscle Nerve 1997;20:1328-30.
Philip J. Young, Patricia M. Day, Jianhua Zhou, et al. A direct interaction
between the survival motor neuron protein and p53 and its relationship to
apinal muscular atrophy. J Biol Chem 2002; 277:2852–9.
Ilanogovan R, Marshall WL, Hua Y. Inhibition of apoptosis by
Z-VAD-fmk in SMN-depleted S2 cell. J Biol Chem 2003;278:30993-9.
Raff MC, et al. Size control: the regulation of cell numbers in animal
development. Cell 1996;86:173-5.
Rietschel M, Rudnik-Schoneborn S, Zerres K, et al. Clinical variability of
autosomal dominant spinal muscular atrophy. J Neurol Sci
1992;107:65-73.
Ripps ME, Huntley GW, Hof PR, Morrison JH, Gordon JW. Transgenic
mice expressing an altered murine superoxide dismutase gene provide an
animal model of amyotrophic lateral sclerosis. Proc Natl Acad Sci
1995;92:689-93.
Rootman DS, Tatton WG, Hay M, et al. Postnatal histogenetic death of
rat forelimb motorneurons. J Comp Neurol 1981;199:17-27.
Roy N, Mahadevan MS, McLean M, et al. The gene for neuronal
apoptosis inhibitory protein is partially deleted in individuals with spinal
muscular atrophy. Cell 1995;80:167-8.
Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide
dismutase gene are associated with familial amyotrophic lateral sclerosis.
Nature 1993;362:59-62.
Russman BS, Iannocone ST, Buncher CR, et al: Spinal muscular atrophy:
new throughts on the pathogenesis and classification schema. J Child
Neurol 1992;7:347-53.
Sandri M, Minetti C, Pedemonte M, Carraro U, et al. Apoptotic
myonuclei in human Duchenne muscular dystrophy. Lab Invest
1998;78:1005-16.
Schrank B, Götz R, Gunnersen JM, et al. Inactivation of the survival
motor neuron gene, a candidate gene for human spinal muscular atrophy,
leads to massive cell death in early mouse embryos. Proc Natl Acad Sci
USA 1997;94:9920-5.
Simic G, Seso-Simic D, Lucassen PJ, Islam A, Krsnik Z, Cviko A, Jelasic
D, Barisic N, Winblad B, Kostovic I, Kruslin B. Ultrastructural analysis
and TUNEL demonstrate motor neuron apoptosis in Werdnig-Hoffmann
disease. J Neuropathol Exp Neurol 2000;59:398-407.
Soler-Botija C, Ferrer I, Gich I, Baiget M, Tizzano EF. Neuronal death is
enhanced and begins during fetal development in type I spinal muscular
atrophy spinal cord. Brain 2002;125:1624-34.
Sophie N, Carmen C, Tony F, Judith M, et al. Spinal muscular atrophy:
recent advances and future prospects. Muscle Nerve 2002;26:4-13.
Suzuki Y, Murakami N, Goto Y, Orimo S, Komiyama A, Kuroiwa Y,
Nonaka I. Apoptotic nuclear degeneration in Marinesco-Sjögren
syndrome. Acta Neuropathol 1997;94:410-5.
Thompson, CB, et al. Apoptosis in the pathogenesis and treatment of
disease. Science 1995;67:1456-62.
Tompkins MM, Basgall EJ, Zamrini E, Hill WD. Apoptotic-like changes
in Lewy-body-associated disorders and normal aging in substantia nigral
neurons. Am J Pathol 1997;150:119-31.
Warner CL, Servidei S, Lange DJ: X-linked spinal muscular atrophy
(Kenneydy''s syndrome) a kindred with hypobetalipoproteinemia. Arch
Neurol 1990;47:1117-20.
Wirth B, Rudnik-Schöneborns, Hahnen E, et al. Prenatal prediction in
families with autosomal recessive proximal spinal muscular atrophy
(5q11.2-q13.3): molecular genetics and clinical experience in 109 cases.
Prenat Diagn 1995;15:407-17.
Yaraghi Z, McLean MD, Surh NRL, et al. A recombination event
occurring within two complex 5q13.1 microsatellite repeat
polymorphisms suggests a telomeric mapping of spinal muscular atrophy.
Hum Genet 1995;96:330-4.
Yan C, Ikezoe K, Nonaka I. Apoptotic muscle fiber degeneration in distal
myopathy with rimmed vacuole. Acta Neuropathol 2001;101:9-16.
Zerres K, Rudnik-Schõneborn S, et al. Natural history in proximal spinal
muscular atrophy: clinical analysis of 445 patients and suggestions for a
modification of existing classification. Arch Neurol 1995;52:518-23.