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研究生:霍其嶸
研究生(外文):Sheridan Ke-wing Fok
論文名稱:Avi-caspase X在瓢體蟲前端再生中的表現量是由經典Wnt/β-catenin訊息傳遞路徑所調控
論文名稱(外文):Avi-caspase X Expression in Anterior Regeneration of Aeolosoma viride Is Regulated by the Wnt/β-Catenin Signaling Pathway
指導教授:陳俊宏陳俊宏引用關係
指導教授(外文):Jiun-Hong Chen
口試委員:郭典翰吳益群蔡素宜
口試委員(外文):Dian-Han KuoYi-Chun WuSu-Yi Tsai
口試日期:2018-01-05
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:71
中文關鍵詞:瓢體蟲再生細胞凋亡胱天蛋白酶Wnt 訊息傳遞路徑
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細胞凋亡是生物體內自發性的一種細胞程序性死亡,其參與生物體內許多重要的生理機制,像是胚胎發生、免疫反應、及神經退形性疾病等,生物體內的細胞凋亡主要是由一群專一性極高的蛋白脢-胱天蛋白酶(caspase)所調控。在渦蟲和非洲爪蟾的實驗中已經發現,caspase在再生的過程中亦扮演極為重要的角色;另一方面,經典Wnt/β-catenin訊息傳遞路徑參與發生過程中許多重要的程序,包含細胞分裂、細胞遷移、體軸建立及組織再生。研究顯示經典Wnt/β-catenin訊息傳遞路徑和胱天蛋白酶可互相調控並且影響生物體的再生。瓢體蟲(Aeolosoma viride)是一種具有極強再生能力的水生環節動物,因此在此篇論文中被用來作為模式生物進行細胞凋亡與經典Wnt/β-catenin訊息傳遞路徑調控再生的研究。
此篇論文的研究結果確認了細胞凋亡會於前端再生早期及晚期出現於再生組織中。三個與細胞凋亡相關的基因被從瓢體蟲身上選殖出來,再經由序列及演化樹的分析確認這三個基因分別為:caspase、Bcl-associated X protein (bax)、B-cell lymphoma extra large(Bcl-xL)。其中caspase跟目前已知caspase構造不盡相同,故命名為:Avi-caspase X。會促進細胞凋亡進行的Avi-caspase X 及Avi-bax均會在再生晚期大量的表現;而具有抑制細胞凋亡功能的Avi-Bcl-xL則是在再生早期大量表現並於24小時後逐漸降低。在使用RNA干擾抑制Avi-caspase X 基因表現量時,再生成功率會大幅的下降,說明了Avi-caspase X 對於瓢體蟲前端再生的重要性。這些結果均顯示細胞凋亡及caspase參與了瓢體蟲前端再生的過程。另一方面,我們發現瓢體蟲前端再生的過程中,Avi-Wnt-4的基因表現量會顯著的上升;以Wnt抑制劑 XAV939抑制Wnt訊息傳遞路徑活性時,會使再生完成率及Avi-caspase 6和新發現的Avi-caspase X的基因表現量都會顯著的下降;Bax的表現量亦會隨著caspase的表現量下降,而抑制性的Bcl-xL則會上升。但是,當Avi-caspase X 的活性被抑制時,Avi-Wnt-4的表現量並沒有產生明顯的變化。因此,推論WNT應該屬於細胞凋亡的訊息傳遞路徑的上游,可共同調控再生的進行。
Many invertebrates possess extraordinary regenerative ability to repair their lost tissues or organs. Apoptotic caspases are required during early stage regeneration in Xenopus laevis and many model organisms. Previous study showed that Aelosoma viride could not complete its anterior regeneration when Avi-caspase 6 was knocked down by RNA interference. However, caspase was not the only factor affecting anterior regeneration in A. viride. The canonical wnt/β-Catenin signaling pathway had been prove to be involved in many biological processes such as cell proliferation, cell migration, carcinogenesis, axis formation, and tissue regeneration. Recent studies in Hydra vulgaris also indicated that wnt/β-Catenin signaling might have promoted apoptosis through a caspase-dependent pathway. In A. viride, expression of Avi-wnt-4 elevated in the anterior region during anterior regeneration, and Wnt antagonist, XAV939, showed a significant inhibitory effect during anterior regeneration.
In this study, I have identified a novel caspase, Avi-caspase X. Its expression was elevated during anterior regeneration, and was confirmed by both in situ hybridization and quantitative real-time polymerase chain reaction (qRT-PCR). In order to identify the relationship between apoptotic caspase and canonical Wnt signaling pathway, inhibitor of Wnt signaling pathway was applied to observed the change in caspase gene expression. Surprisingly, expression of both Avi-caspase X and Avi-caspase 6 decreased significantly in XAV939 treated group. The Bcl-2 protein family is notable for their regulation of apoptosis. In order to rule out the possibilities of genetic cross talk, two target genes, Bcl-2 associated X protein (apoptosis regulator Bax) and B-cell lymphoma-extra-large (Bcl-xL) from the Bcl-2 family were identified and examined. The gene expression level of Avi-Bax and Avi-Bcl-xL fluctuated during anterior regeneration, and seems to be negatively correlated to each other. Under the treatment with XAV939, the expression level of Avi-Bax decreased along with an increase in gene expression level of Avi-Bcl-xL. Finally, RNA interference (RNAi) of Avi-caspase X was performed by siRNA, further confirmed the relationship between Wnt signaling pathway and apoptotic caspase.
Contents
致謝 iii
中文摘要 vii
Abstract viii
Introduction 1
Regeneration 1
Caspase 2
Apoptosis 3
Roles of caspase in the process of development and regeneration 4
Canonical Wnt signaling pathway 5
Aeolosoma viride 6
Material Methods 8
Aeolosoma viride 8
RNA Extraction 8
Reverse Transcription 9
Real time quantitative polymerase chain reaction (RT-qPCR) 10
in situ hybridization 10
TUNEL assay 12
dsRNA 13
Microinjection 14
Statistical analysis 14
Results 15
The number of apoptotic cells during anterior regeneration 15
Sequencing of Avi-caspase X, Avi-Bax, and Avi-Bcl-xL 15
Gene expression of Avi-caspase X, Avi-caspase 6, Avi-Bax, and Avi-Bcl-xL during anterior regeneration 18
Location of gene expression of Avi-caspases at the blastema during anterior regeneration 19
Inhibition of Avi-caspase X by dsRNA 20
Inhibitory effect of XAV939 on anterior regeneration in A. viride 21
The influence of XAV939 on apoptosis during regeneration 21
Avi-caspase X dsRNA showed no effect on the gene expression of Avi-Wnt-4 22
Discussion 24
Apoptosis and regeneration 24
Identification of a novel caspase, Avi-caspase X 24
Avi-caspase X expression is critical during anterior regeneration 25
Avi-caspase X and Avi-caspase 6 26
Relationship between apoptotic caspase and canonical Wnt signaling pathway 27
Compensatory proliferation in A. viride 28
Reference 29
Tables 35
Table 1. primers used for cloning apoptotic related genes 35
Table 2. primers used for the RNA probes 35
Table 3. primers used for qPCR 36
Table 4. primers used for the synthesis of dsRNA 36
Figures 37
Figure 1. Detection of apoptotic cells during anterior regeneration 39
Figure 2. Sequence of Avi-caspase X, Avi-Bax, Avi-Bcl-xL 46
Figure 3. Phylogenetic tree for Avi-caspases 48
Figure 4. Phylogenetic tree for Avi-Bax and Avi-Bcl-xL 50
Figure 5. Relative gene expression of Avi-caspases during anterior regeneration 52
Figure 6. Relative gene expression of apoptotic related genes during anterior regeneration 54
Figure 7. Localization of Avi-caspases during anterior regeneration 57
Figure 8. Knock-down experiment of Avi-caspase X by dsRNA 60
Figure 9. The inhibitory effect of XAV939 on anterior regeneration of A. viride. 62
Figure 10. The inhibitory effect of XAV939 on anterior regeneration of A. viride. 64
Figure 11. Gene expression of Avi-caspases, Avi-Bax, and Avi-Bcl-xL decreased under XAV939 treatment 66
Figure 12. Avi-caspase X dsRNA treatment showed no significant effect on gene expression of Avi-Wnt-4 68
Figure 13. Apoptotic signals increased after XAV939 treatment 71
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