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研究生:莊雁茹
研究生(外文):Yen-Ju Chuang
論文名稱:第I型脊髓肌肉萎縮症小鼠之脊髓運動神經元細胞凋亡研究
論文名稱(外文):The Study of the Apoptotic Spinal Motorneuron in Type I Spinal Muscular Atrophy Mice
指導教授:鐘育志鐘育志引用關係
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:59
中文關鍵詞:細胞凋亡脊髓運動神經元脊髓肌肉萎縮症
外文關鍵詞:apoptosisspinal motorneuronspinal muscular atrophy
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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
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