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

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。

關鍵字

細胞凋亡；脊髓運動神經元；脊髓肌肉萎縮症

並列摘要

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.