臺灣博碩士論文加值系統

活化素(activin)屬於轉化生長因子-β (Transforming Growth Factor-β)，已知與傷口癒合、發炎反應、細胞分裂及細胞凋亡等有密切關係。過去的研究發現，activin在老鼠的傷口癒合和斑馬魚的尾鰭再生都扮演著重要的腳色，當activin被抑制的時候，老鼠的傷口無法正常的癒合，還有斑馬魚的尾鰭也無法再生。再者，近期研究發現活化素的拮抗分子follistatin在渦蟲再生過程中的角色非常重要，因此，本研究想確認activin在無脊椎動物中對於再生的重要性。我們選用的模式動物為瓢體蟲Aeolosoma viride，牠是一種水生的環節動物，能在五天再生完成失去的頭部。本篇研究中發現，在A. viride頭部再生過程中，activin會大量的表現，activin receptor會集中表現在A. viride傷口的位置。接著，以activin/TGF-β的抑制劑 SB505124處理再生中的A. viride時，傷口癒合無法正常進行，且再生會被抑制。綜合以上實驗結果，我們推測activin/TGF-β在A. viride中可能透過傷口表皮癒合來影響再生。

Activin, a TGFβ superfamily protein, is known to regulate cell proliferation, inflammation, apoptosis, regeneration, and wound healing. Previous researches demonstrated that activin is necessary for wound healing in mice and fin regeneration in zebrafish. Recently, follistatin has been linked to regeneration in planarians. In this research, we investigated the roles of activin in the wound healing of a small fresh water annelid, Aeolosoma viride. A.viride can fully regenerate the head within 5 days after amputation. The results showed activin expression is upregulated during regeneration and activin receptor is densely expressed at the wound site and the blastema. Furthermore, A. viride treated with a chemical inhibitor of activin/TGF-β, SB 505124, obviously inhibited wound healing and impaired regeneration. Therefore, we infer that activin/TGF-β signaling might mediate wound healing to affect anterior regeneration in A. viride.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
Introduction 1
Regeneration 1
Activin/TGF-β Signaling 2
Activin/TGF-β Signaling and Regeneration 3
Aeolosoma viride and Regeneration Research 4
Aim 5
Materials and Methods 6
Aeolosoma viride 6
Irradiation 6
RNA Extraction 6
Reverse Transcription 7
Gene Cloning 7
Synthesis of DIG-labeled Probes 8
In situ hybridization 8
Quantitative real-time PCR 9
Western Blot 9
Immunofluorescence 10
Results 12
Regeneration of Aeolosoma viride 12
Sequences of Avi-activin, Avi-follistatin and Avi-activin receptor 12
Activin/TGF-β expression profiles during early anterior regeneration 13
Activin/TGF-β expression in the epithelium of A. viride 13
Inhibition of activin/TGF-β signaling impairs regeneration 14
Inhibition of activin/TGF-β signaling disables wound healing 16
Discussion 17
References 21
Tables, Images and Figures 27
Table 1. Primers Used for cDNA Identification 27
Table 2. Primers Used for qRT-PCR Identification 28
Image 1. Map of primers and probes on complete Avi-activin sequence. 28
Image 2. Map of primers and probes on complete Avi-follistatin sequence. 28
Image 3. Map of primers and probes on complete Avi-activin receptor sequence. 29
Figure 1. Aeolosoma viride. 30
Figure 2. Sequence of Avi-activin. 32
Figure 3. Sequence of Avi-follistatin. 34
Figure 4. Sequence of Avi-activin receptor. 36
Figure 5. Expression profiles of Avi-activin, Avi-follistatin and Avi-activin receptor during early regeneration. 38
Figure 6. Expression patterns of Avi-activin, Avi-follistatin and Avi-activin receptor. 39
Figure 7. Immunohistochemistry of Activin in intact and regenerating A. viride. (A) 40
Figure 8. Effects of SB-505124 treatment on the regeneration of A. viride. 41
Figure 9. Cell proliferation in the regenerating tissue of A. viride with or without SB505124 treatment. 43
Figure 10. Effects of SB505124 treatment on wound closure. 45
Figure 11. Radiation effects on wound closure in A. viride. 46

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