臺灣博碩士論文加值系統

在動物界中，許多動物都能透過再生修補受損部位。然而對於多數動物而言，再生能力是會隨著年紀增長而隨之下降的。比較年輕與年長的生物，許多異時性基因，因為會在特定時間表現並隨著年紀的增加而隨之下降而被視為可能造成此現象的候選目標。Lin28，是一個 RNA 的結合蛋白，最早在線蟲體內發現的。在多數的動物中，lin28 也是在成體之前大量表現，進入成體之後快速下降。不僅如此，前人研究中也發現高度表現lin28 能夠增加斑馬魚以及成體老鼠的再生能力。而為了要了解再生與老化之間的關係以及 Lin28 所扮演的角色，我們引進了瓢體蟲作為我們的實驗動物。瓢體蟲是一種淡水生的環節動物，具有很強的再生能力以及很短的壽命。將它頭部切除後，瓢體蟲能在五天的時間內完成再生。另一方面，前人研究也指出它的平均壽命約 40 天，是非常適合作為再生與老化的研究。本篇研究中，我們選殖瓢體蟲的lin28 序列，並且確認 Lin28 可透過促進細胞的重新規劃以及增生來幫助前端再生的進行。而與年輕蟲體前端進行再生時比較，發現在年長蟲體中lin28 的基因表現量依舊會上升，且 Lin28 所調控的細胞重新規劃以及增生都無看到顯著的差異，由此可知在瓢體蟲中年長蟲體再生能力下降的現象，可能不是由再生前期的相關事件如細胞重新規劃以及增生所造成的，可能是與後期的組織成型有較大的關係。

In animal kingdom, many animals can regenerate or repair the lost parts after injury. However, the regeneration capacity of most animals declines in aging. The expression levels of many heterochronic genes decline sharply in older animals, and these genes are regarded as the candidates to study the decline of regenerative ability in aging. Lin28 (cell lineage abnormal 28) is an RNA binding protein first identified in C. elegans. In many animals, lin28 highly expresses before adulthood, and its expression decreases dramatically in the adult. Nevertheless, previous studies have shown that upregulation of lin28 could enhance the regeneration capacity in zebra fish and adult mice. To characterize the relationship between aging and regeneration, as well as the roles of Lin28 in this process, a new animal model, Aeolosoma viride was introduced. A. viride is a fresh annelid with an outstanding regenerative ability and short lifespan. After anterior amputation, the worm can completely regenerate within five days. On the other hand, previous studies had shown that the mean lifespan of the worm is around 40 days. Thus, it is suitable for the study of regeneration and aging. In this study, I found that Avi-lin28, an ortholog of lin28 in A. viride, increased significantly in the regenerating tissues. Also, Avi-Lin28 was confirmed to participate in anterior regeneration through promoting cell reprogramming and proliferation. However, compared to the young worms, Avi-lin28 expression level did not decrease during anterior regeneration in the old worms. In addition, cell de-differentiated and cell proliferation did not decline significantly during anterior regeneration in the old worms. Thus, based on the results above, it is inferred that the decline of regeneration capacity in aged A. viride is not due to down regulation of cell de-differentiation and proliferation. Rather, it results from abbreviated tissue patterning, which occurs during a later stage of regeneration in A. viride.

致謝 ii
Abstract iv
中文摘要 vi
Introduction 1
Regeneration 1
Regeneration capacity through lifespan 2
Mechanism of Lin28 3
Roles of Lin28 in the processes of regeneration and aging 4
Experimental animal model- Aeolosoma viride 5
Aims: 6
Material and method 7
Experimental animals and sample preparation 7
Regeneration experiment 7
Gene cloning and sequence analysis 8
RNA exaction and quantitative real-time RT-PCR (qRT-PCR) 8
EdU labeling 9
Whole-mount in situ hybridization (WISH) 9
Northern blotting 11
Statistical analysis 12
Results 13
Identification of lin28 sequence in A. viride 13
Relative gene expression of Avi-lin28 at anterior regeneration tissues during anterior regeneration 13
Spatial distribution of Avi-lin28 mRNAduring anterior regeneration 14
Change of matureAvi-let-7 expression at 48 hpa during anterior regeneration 14
The inhibitory influence of Lin28 1632 for A. viride during anterior regeneration 15
Inhibitory effect of Lin28 1632 on signal of Avi-PIWI and cell proliferation during anterior regeneration 17
Lifespan of A. viride 17
Gene expression of Avi-lin28 during different stages of aging in A. viride 18
Signal of Avi-PIWI and cell proliferation at 48 hpa during anterior regeneration in worm of different ages 19
Discussion 21
Reference 26
Tables 35
Table 1. primers used for cloning Avi-lin28 35
Table 2. primers used for the synthesis RNA probe 35
Table 3. primers used for qRT-PCR 35
Table 4. primers used for synthesis of oligo probe for Northern blotting 35
Figure 36
Figure 1. External morphology of Aeolosoma viride and sequence of Avi-lin28. 37
Figure 2. Phylogenetic tree of Lin28. 39
Figure 3. Relative gene expression of Avi-lin28 during anterior regeneration. 41
Figure 4. Spatial distribution of Avi-lin28 during anterior regeneration. 43
Figure 5. Change of Avi-let-7 expression at 48 hpa during anterior regeneration. 46
Figure 6. The inhibitory influence of Lin28 1632 on anterior regeneration of A. viride. 48
Figure 7. The inhibitory effect of Lin28 1632 on Avi-PIWI gene expression and cell proliferation during anterior regeneration in A. viride. 50
Figure 8. Lifespan of A. viride. 52
Figure 9. Avi-lin28 expression level of different age worms. 54
Figure 10. Relative Avi-lin28 expression level at 48 hpa in 2 week, 4 week, 6 week and 8 week worms. 56
Figure 11. The signal of Avi-PIWI at 48 hpa during anterior regeneration in different age of worms. 58
Figure 12. The comparison in signal of cell proliferation at 48 hpa during anterior regeneration in different ages of worms. 60

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