- 學位論文
神經膠細胞株衍生之神經營養分子在培養細胞及脊髓肌肉萎縮症小鼠中之表現
Glial cell line-derived neurotrophic factor in cultured cells and spinal muscular atrophy mice
摘要
脊髓肌肉萎縮症(SMA)是由脊髓前角細胞之退化所造成，其發生率約為一萬分之一。臨床上根據疾病嚴重程度及發病時間可分為三型：第I型、第II型及第III型。此三型皆由存活運動神經元基因1 (survival motor neuron 1 gene, SMN1)之缺損或突變所致，但同染色體上靠近中心節，與SMN1 gene非常類似之存活運動神經元基因2 (SMN2 gene)則無缺損。 神經膠細胞株衍生之神經營養分子(glial cell line-derived neurotrophic factor, GDNF)在帕金森氏症中之含多巴胺之神經元以及脊髓側索硬化症中之運動神經元，已被證實具有神經保護及修復效果，為了研究此分子在同是運動神經元退化疾患之脊髓肌肉萎縮症中所扮演的角色，我們利用反轉錄定量多聚合酶連鎖反應 (real-time reverse transcription polymerase chain reaction, RT-PCR)分析三天大、一個月大、一年大之野生小鼠及SMA type 3小鼠不同組織中GDNF mRNA之表現量。我們亦利用微小神經膠細胞(BV-2 cell)分別與運動神經元細胞(NSC-34 cell)及減量SMN蛋白質運動神經元細胞(SMN-knockdown NSC-34 cell)共同培養，以了解在SMN蛋白質缺少的環境下是否會刺激BV-2 cell製造較多的GDNF。最後，我們以不同濃度GDNF加入SMN-knockdown NSC-34 cell，並使用細胞增殖測定(proliferation assay)及軸突長度測量(neurite length measurement)來評估GDNF的神經保護作用。 我們的研究結果顯示，在新生小鼠及一個月大小鼠階段，SMA type 3 小鼠的GDNF 的mRNA表現量是比野生型小鼠要來得多，但到了成鼠階段，卻似乎呈現相反的結果。在細胞培養中我們發現，當BV-2與SMN-KD NSC-34共同培養時會表現較高量的GDNF，此結果表示SMN蛋白質的減少可能會刺激增加GDNF的表現。而在proliferation assay及neurite length measurement所得到的結果雖然並沒有太顯著的差異，但仍建議利用動物模型進行更進一步的評估。本研究希望針對在SMA中GDNF所伴演的角色提供全新的見解，並進而發展新的治療對策。
並列摘要
Spinal muscular atrophy (SMA) is characterized by degeneration of the anterior horn cells of the spinal cord, which affects 1/10,000 live births. There are three main types of SMA types I, II and III, based on the severity of the disease and age of onset, and all three types is associated with deletions or mutations in the survival motor neuron 1 (SMN1) gene, which has been designated SMN1 to distinguish from its centromeric homologue, SMN2. Glial cell line-derived neurotrophic factor (GDNF) has showed its promising effects in protecting and repairing dopamine-containing neurons, which degenerate in Parkinson's disease, and motoneurons, which die in amyotrophic lateral sclerosis. To investigate the role of GDNF in spinal muscular atrophy (SMA), we analyze GDNF mRNA expression by real-time reverse transcription polymerase chain reaction (RT-PCR) in different tissues from 3-day-old, one-month-old and one-year-old wild type and SMA type 3 mice. Co-cultures of microglial cell (BV-2) and motoneuron cell (NSC-34)/SMN knockdown motoneuron cell (SMN-KD NSC-34; 50% knockdown) were used to detect the GDNF expression under SMN-deficient condition. We also treated the SMN-KD NSC-34 cell by different concentrations of GDNF and proliferation assay and neurite length measurement were applied for evaluating neuroprotective effect. Our study shows GDNF expression is higher in SMA mice than wild-type mice at newborn and one-month age, but seems opposite in adult age. Higher GDNF mRNA expression when BV-2 cell co-cultured with SMN-KD NSC-34 cell, may indicate SMN deficiency would stimulate GDNF expression. The proliferation assay and neurite length measurement show modest results but further animal model assessment is still suggested. This is a new insight to study the role of GDNF in SMA and may provide new therapeutic strategy of SMA.
參考文獻
延伸閱讀
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