Abstract
摘要:脊髓性肌肉萎縮症 (Spinal muscular atophy, SMA) 為主要造成嬰兒死亡的隱性遺傳疾病之一,其發生原因主要係為運動神經元存活蛋白(survival motor neuron, SMN )功能不全所致。目前脊髓性肌肉萎縮症仍無有效療法,透過體細胞核移置(somatic cell nuclear transfer,SCNT ) 技術產製病人特異性胚幹細胞(patient-specific embryonic stem cells, ntESCs)及骨髓間葉幹細胞(mesenchymal stem cells )所提供之自體(autologus )來源細胞,於未來提供此等神經退化性疾病及其他人類各種疾病治療與再生醫學應用帶來無限希望。
運動神經元存活蛋白含量已知與脊髓性肌肉萎縮症的嚴重程度具有極大之相關性。根據研究證實SMN於小型核蛋白(small nuclear ribouncleoprotein, snRNPs)之生合成及RNA 接合(RNA splicing)過程扮演關鍵性的角色。而目前研究報告亦指出,RNA 接合於幹細胞全能性與分化扮演重要性的角色。在果蠅模式之最新實驗研究顯示於體內多種組織中,未分化之細胞具有高含量之SMN蛋白(如卵巢中之生殖細胞),反之,已分化細胞之SMN蛋白卻僅保有極低之含量。吾等因此假設 SMN 可能透過參與RNA 接合過程,調節未分化幹細胞之自我更新或分化的能力。來自本整合型計畫所提供之骨髓間葉幹細胞及吾等近期研究所建立之體細胞核移與產製特異性胚幹細胞關鍵性技術,配合一系列分子生物技術 (包括knockdown and overexpression等)及微陣列分析(microarray)方法探討運動神經元存活蛋白的消長對於全能性胚幹細胞、多能性間葉幹細胞及其他高度分化體細胞的影響,將提供獨特之平台探討RNA 接合於幹細胞學所扮演的角色,進而可能解釋高度分化細胞與導致運動神經元存活蛋白缺失之間的關聯性。而透過本子計畫體細胞複製技術所建立之疾病模型的特殊自體來源之胚幹細胞,亦可提供本整合型計畫之其他相關子計畫進行後續幹細胞相關研究。
故本三年期整合型之子計畫三擬:1)建立運動神經元存活蛋白在小鼠與豬的不同組織與各分化程度細胞的分布情形,著床前胚胎、全能性幹細胞、纖維母細胞及各體組織將做為進行探討運動神經元存活蛋白體內衡定(homeostasis)之基本平台; 2)小鼠脊髓性肌肉萎縮症幹細胞(SMA-ntESCs )特性模式之建立:運動神經元存活蛋白的消長與胚胎幹細胞之全能性及其神經細胞分化的關聯性;3)運動神經元存活蛋白於調節豬骨髓間葉幹細胞增值與分化角色之研究。藉本計劃之執行,除進一步深入了解運動神經元存活蛋白缺失程度與神經退化或幹細胞特性及其分化調控機制之關聯性外,其結果並可提供本整合型計畫相關子計畫研究幹細胞特性維持及其分化與否之重要參考;於臨床醫學應用上,將提供重要依據發展充分具功能性之種子細胞來源,冀未來於脊髓性肌肉萎縮症或其他人類疾病治療,藥物篩選與再生醫學上,邁入另一新的里程碑。
Abstract: Spinal muscular atrophy (SMA) is leading genetic cause of infant death and one of the most common autosomal recessive diseases, which is caused by deficiency of the survival motor neuron (SMN) proteins. So far, therapies in this human neurodegenerative disease are remained ineffective. Somatic cell nuclear transfer to derive patient-specific embryonic stem cells (ntESCs, known as therapeutic cloning) and mesenchymal stem cells (MSCs) derived from bone marrow represent wild therapeutic applications and considered as most promising autologus resource for repair tissues in degenerative diseases, such as Parkinson’s disease or SMA.
The level of SMN protein is highly relative with severity of SMA. It has been demonstrated that SMN plays an essential role for small nuclear ribouncleoprotein (snRNPs) biogenesis and RNA splicing. Recent studies in Drosophila showed that SMN is evident in multiple tissues with the highest levels in undifferentiated stem cells and lowest in the differentiated cells. The RNA splicing has also been recognized as a critical factor for stem cell pluripotency and differentiation. Therefore, we hypothesis that SMN might play an important role through RNA splicing to regulate function of self-renew in undifferentiated stem cells. Currently, our lab has well-established disease-ntESC lines and a plentiful amount of MSCs resources provide from current integrated program in Subproject 1, 2, and 5. Through loss-of-function or gain-of-function strategy by knockdown or overexpression of SMN in ESC, MSCs and somatic cells may provide a valuable model to link RNA splicing and stem cell biology and potentially explain how highly differentiated cells can become vulnerable to SMN reduction.
Therefore, in this study, we aim to understand the functions and signaling pathways of SMN in maintenance and differentiation of stem cells in order to provide a basis for stem cell therapy for human neurodegenerative disorders. First, we will focus on 1) characterize SMN expression pattern in differentiated and undifferentiated cells in mouse and porcine. Pre-implantation embryos, ESCs, fibroblast cells and tissues will use as basic platform to study homeostasis of SMN. Next, 2) establish a “stemness” model for SMA in mouse: SMA-ntESCs pluripotency and neuron differentiation potential following SMN protein knockdown or overexpression. Finally, 3) study the role of SMN in regulation of proliferation and differentiation in porcine MSCs, and differentiate MSCs into motor neuron-like cells for potential resource of stem cell therapy in SMA. Success of our research project would help us to understand not only the cellular mechanism of SMA but also provide a potential therapeutic resource for human neurodegenerative diseases.
Keyword(s)
幹細胞特性
細胞分化
運動神經元存活蛋白
脊髓性肌肉萎縮症
動物模式
stem cells
differentiation
survival motor neuron proteins (SMN)
spinal muscular atrophy (SMA)
animal model