- 學位論文
細胞凋亡基因 caspase-3/7 於瓢體蟲頭部再生中所扮演的角色
The Roles of Apoptotic Gene caspase-3/7 on Anterior Regeneration in Aeolosoma viride
摘要
再生（Regeneration）是近年來相當熱門的研究，當動物受傷後，失去部分組織或器官，經過細胞增生及分化過程以進行修復失去部分組織或器官，使個體回復至正常生理功能。過去研究再生的模式動物，如：脊椎動物的蠑螈和斑馬魚及無脊椎動物的渦蟲和水螅，這些模式動物皆擁有良好的再生能力，可重新長出新的組織或器官。 在過去研究指出當動物受傷後，傷口處的細胞先歷經死亡，將死亡細胞移除後，經過細胞增生以修復受損的部位，完成再生過程（Regeneration）。本研究想探討受傷處的細胞死亡現象，細胞凋亡（Apoptosis），又名為程序性細胞死亡（programmed cell death），可讓受損的細胞自動進入死亡程序，以維持生物體內部的恆定，如：器官形態修飾、動物形體變態、癌細胞的移除等過程。在 apoptotic pathway 中，細胞凋亡基因: caspase-3 或 caspase-7 在細胞凋亡過程，扮演著關鍵性的角色。本研究想探究 apoptosis 與再生之間的關係，確認 caspase-3 或 caspase-7 在無脊椎動物再生過程中的影響力。 本研究的實驗物種瓢體蟲（Aeolosoma viride）為淡水生環節動物，其具有相當良好的再生能力，可於切除頭端的五天後再生出完整的頭端。在本研究中，首先 clone到一個疑似 caspase-3 及 caspase-7 的基因，故暫定名為 caspase-3/7。除了發現此基因確實參與再生過程外，值得注意的是 Avi-cas-3/7 於再生中的表現為先升後降，接著再升，最後又下降的趨勢。推測 Avi-cas-3/7 於再生過程中先被活化後關閉，接著再活化，最後停止反應。第一波提升為了將受損細胞移除，而第二波提升則是為了將細胞增生過程中多餘的細胞移除，當所有任務完成後即消失。此外，為了瞭解 Avi-cas-3/7 對於再生過程的重要性，以 RNAi 抑制 Avi-cas-3/7 的表現後，再進行截頭處理。在本研究中發現當 Avi-cas-3/7 受到抑制時，將影響後續頭部再生，造成頭部無法再生。 綜合上述實驗結果，推測於再生過程中，Avi-cas-3/7 確實參與其中之外，分別於再生前期（移除受損細胞）和再生後期（移除多餘的增生細胞）具有高度表現量。除此之外，抑制 Avi-cas-3/7 後，無法進行頭部再生。顯示 Avi-cas-3/7 具有調控 A. viride 頭部再生的能力。
並列摘要
In recent years, regeneration is a hot issue. When animal tissues or organs are damaged or lost, the processes of cell proliferation and differentiation can repair the damaged tissues or organs helping the animal to survive. Several animal species have been used to investigate regeneration, including hydra, planarian, salamander and zebrafish. Previous studies showed that when animals are wounded, the programmed cell death, apoptosis, would happen at the wound site before regeneration occurred. It is well known that apoptosis is a homeostatic mechanism for removing the damaged cells. In apoptosis, caspase-3 and caspase-7 are required functional genes to kill cells. Although the relationship between apoptosis and regeneration had been documented, their linkage was not clearly demonstrated. Aeolosoma viride is a fresh water annelid, which has been used as a model organism for studying regeneration recently. When its anterior region was amputated, the whole anterior part could completely regenerate within five days. In this study, a caspase gene of A.viride was cloned, and its nucleotide sequence is similar to caspase-3 and caspase-7. Therefore, it was named Avi-cas-3/7. The results indicated that Avi-cas-3/7 participated with anterior regeneration in A. viride. It showed a specific expressed pattern during regenerative process. Its gene expressions had increased and decreased twice before the end of regeneration. This result was inferred that the first peak of Avi-cas-3/7 expression might involve in removing the damaged cells, and the second peak might relate to pattern formation of anterior region in which the extra cells were removed during regeneration. Furthermore, Avi-cas-3/7 expression was knocked down by RNA interference (RNAi), and anterior regeneration was inhibited. This result indicated that Avi-cas-3/7 has a crucial role in regeneration in A. viride. In summary, apoptotic gene, Avi-cas-3/7 is actually involved in the anterior regeneration in A. viride.