於蠑螈手臂再生時尋找其差異性表達基因和證明持續性肌肉去分化

Identification of differentially expressed genes and demonstration of long-duration muscle dedifferentiation during limb regeneration in axolotls

巫承翰(Cheng-Han Wu)

指導教授 ： 李宣書

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.02376

摘要

有尾目兩棲動物具有非凡的肢體再生能力，當墨西哥蠑螈肢體被截斷後，將會啟動傷口癒合反應，接著形成原基組織並且再生出全新的肢體，在肢體再生的起始階段，原基細胞是由神經和頂端上皮蓋所誘導產生，神經在肢體再生中的作用一直以來都被頻繁的研究，雖然目前對於蠑螈肢體再生的神經依賴性已經有一定程度的了解，但是在探討神經依賴性底層機制的過程中，大規模系統化的探索還是有其必要性；在本篇研究中，高通量定序技術被用來比對再生肢體和去神經肢體的大量轉錄體，實驗的結果揭露多種原基組織和神經依賴性相關的生物程序，在將來一定能夠助益蠑螈肢體再生相關之研究；此外，我們藉由觀察蠑螈手臂再生時肌肉末端Pax7轉錄體的表現情況，對於蠑螈從原基中期到分化晚期的持續性去分化這現象，提供了扎實的證據，並且利用活體擴散張量影像技術和免疫染色實驗觀察到新生肌肉和殘餘肌肉之間具有一個接合性縫隙，綜合來看，由以上研究結果顯示出殘餘肌肉的持續性去分化現象，使得殘餘肌肉細胞保持較未分化的狀態，將能夠幫助新、殘餘肌肉組織準確的接合；這些實驗結果也許在不遠的將來，能夠幫助了解肢體再生的機制和協助再生醫學的發展。

關鍵字

原基 ； 肢體再生 ； 神經依賴性 ； 墨西哥蠑螈 ； 去分化 ； 肌肉 ； 擴散張量影像 ； 再生醫學

並列摘要

Urodele amphibians have an extraordinary ability to regenerate lost limbs. Amputation of the limb of axolotl causes an initial wound healing response followed by blastema formation and the regeneration of the new limb. In initial stage of limb regeneration, blastemal cells are induced by nerves and the apical epithelial cap. Nerve functions have long been the focus of limb regeneration studies. Although considerable progress has been made in inquiring into the nerve-dependency of salamander, a broader systemic perspective is still needed to investigate the underlying mechanisms upon the presence of a nerve. In this study, a high-throughput sequencing approach was used to compare transcript abundance among regenerating limbs, and limbs denervated at the time of amputation. This result addressed the biological processes which are associated with blastemal formation and nerve-dependency, and will greatly assist future research of limb regeneration in axolotl. Furthermore, I added evidence that dedifferentiation persisted from mid-bud to late differentiation stage by showing expression of Pax7 transcripts in the remnant muscle fiber ends. Moreover, in vivo diffusion tensor imaging and stained tissue sections indicate a gap between the regenerating and parental muscles. Taken together, the present data suggested that prolonged dedifferentiation at remnant muscle ends might facilitate accurate connection between regenerating and parental muscle fibers due to their immature status. Additionally, these results may assist in the understanding of limb regeneration, ultimately facilitating the development of regenerative medicine in mammals.

並列關鍵字

blastemal ； limb regeneration ； nerve-dependency ； axolotl ； dedifferentiation ； muscle ； diffusion tensor imaging ； regenerative medicine

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