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標題: | 探討DjAgo2蛋白在渦蟲再生中所扮演的角色 Study on the role of Argonaute protein DjAgo2 in planarian regeneration |
作者: | Chi-Kai Chang 張智凱 |
指導教授: | 朱家瑩 |
關鍵字: | 渦蟲,再生,成體幹細胞, planarian,Ago2,neoblast,stem cell,regeneration, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 渦蟲是一種具有再生能力的扁形動物,能由身體一部分片段再生出包含腦部的完整個體。渦蟲的再生能力建立在一群多功能成體幹細胞(neoblast)身上,他們可以進行複製以及分化來替換身體所有的組織,而且這群細胞對γ-射線很敏感。Dugesia japonica(東洋渦蟲)的DjAgo2蛋白是人類Ago2的同源蛋白質,在人類細胞上的研究顯示hsAgo2是miRNA和siRNA路徑中的關鍵因子,具有調控基因表現的能力。本研究中,我們首先選殖DjAgo2之完整全長cDNA並構築與YFP蛋白接合之重組蛋白表現載體,將其表現在HeLa cell中。在螢光顯微鏡下觀察發現,DjAgo2會和人類的P-body組成物RCK等聚集於細胞質內,這現象和hsAgo2的表現很類似。藉由整體原位雜交的實驗,發現Djago2高度表現在渦蟲的頭部和背中線上。渦蟲尾部再生的實驗中顯示在blastema以及post-blastema兩區Djago2的表現量在再生時會上升,而RT-qPCR的結果也顯示其表現量在再生第三天的blastema和post-blastema中,分別高出平常的五倍和兩倍。藉由FACS來分離neoblast和已分化細胞,發現Djago2在成體幹細胞中的表現量較高,而且γ-射線會使得部分表現Djago2的細胞消失。最後,用Djago2 dsRNA來餵食渦蟲以抑制蟲體中Djago2的表現,渦蟲會失去再生的能力而且會喪失組織恆定性。RT-qPCR的結果顯示表現在幹細胞 (DjpiwiA)以及合成DNA (Djpcna) 的基因表現量在抑制Djago2後會下降。而且,M phase的細胞減少以及G2 phase的細胞增加顯示抑制Djago2會使得成體幹細胞的細胞周期停留在G2 phase而無法進行有絲分裂。同時,也觀察到部分渦蟲出現分化受到限制的現象。我們的實驗結果顯示,Djago2在渦蟲成體幹細胞的細胞複製、自我更新以及分化時都被需要的,可以藉由對於成體幹細胞的調控來影響渦蟲的再生能力。 Planarians are capable of regenerating almost every part of their body, including the brain. Regeneration in planarians is mediated by a group of pluripotent adult somatic stem cells, called neoblasts, which can proliferate and differentiate to replace all tissues. These cells are sensitive to γ-irradiation. DjAgo2 of Dugesia japonica is a homolog of hsAgo2, the key factor that regulates gene expression in miRNA and siRNA pathways. In the study, DjAgo2 was cloned, and YFP-tagged DjAgo2 was expressed in HeLa cells. DjAgo2 accumulates at specific foci that contain the P-body component, RCK. This pattern is similar to what we observed with hsAgo2. By whole-mount in situ hybridization (WISH), Djago2 was found to be highly expressed in regions of the brain and the middle dorsal line. Djago2 expression was increased in the blastema and post-blastema during regeneration. RT-qPCR results confirmed that respective Djago2 mRNA expression levels in the blastema and post-blastema were 5 and 2-times higher than that in the control at 3 days post-amputation. WISH data showed that a portion of Djago2-positive cells were depleted by γ-irradiation. We next examined Djago2 expression levels in neoblasts isolated by FACS. Our RT-qPCR data showed that the Djago2 level was higher in neoblasts than that in differentiated cells. Finally, Djago2 was silenced by feeding planarians Djago2 dsRNA. Depletion of Djago2 resulted in loss of the regeneration capacity and defects in tissue homeostasis in planarians. Djago2 silencing reduced levels of DjpiwiA, Djpcna, and DjpiwiC. Our data also showed that the ratio of M-phase cells was decreased and the ratio of G2-phase cells was increased in Djago2-silenced animals. This suggests that Djago2 silencing blocked the mitosis of neoblasts. Differentiation restriction was also observed in Djago2-silenced animals. Our data indicate that Djago2 is required for neoblast proliferation, and differentiation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6902 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 動物學研究所 |
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