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研究生:董鴻楠
研究生(外文):Hung-Nan Tung
論文名稱:斑馬魚p73基因的選殖及表現分析
論文名稱(外文):cloning and expression study of the zebrafish p73 gene.
指導教授:陳凌雲陳凌雲引用關係潘惠錦
指導教授(外文):Chen L YPan Hui Chi
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:73
中文關鍵詞:斑馬魚
外文關鍵詞:Zebrafishp73
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在人類腫瘤中,p53是最常突變的一種腫瘤抑制蛋白。最近,與p53相似的p73和p63也在人類以及老鼠中被分離出來。但是經由研究發現,p73和p63並不是腫瘤抑制蛋白,而是在發育上扮演重要角色。在此研究中,我們利用RT-PCR和3’ RACE方法從卵巢RNA選殖出斑馬魚的p73 cDNA。由四段重疊的片段定序所得到的cDNA包含一個ORF (1923 bp) 以及5’端和3’端的不轉譯區 (37 bp 及438 bp)。此ORF可轉譯出640個胺基酸,經由比對結果發現,斑馬魚的p73胺基酸與其它物種的相似度為:與人 (Homo sapiens)為71﹪, 與猿猴 (Cercopithecus aethiops)為70%,與老鼠 (Mus musculus)為71%,與鯰魚 (Barbul barbus)為95%, 與蝸牛 (Mya arenaria)為32%。由RT-PCR結果得知,p73在皮膚、腦、魚鰭、卵巢、睪丸有較多表現,在脾臟和肝臟也有微量表現,但在其他組織中則偵測不到表現。p73 RNA在受精3小時後即出現,但量不多。在6、25、32、48、72以及120小時都可偵測得到表現。此外,利用全體原位雜交及免疫化學染色方法,發現p73也在頭部的嗅球及生殖器官的特定細胞上有表現。這些與p53和p63差異的表現,顯示p73,p53和p63可能具有屬於它們專一的功能和角色。

The tumor suppressor p53 is the most frequently mutated protein in human cancers. Recently, homologues of p53, such as p73 and p63, were isolated in human and mice. Instead of acting as tumor suppressors, these proteins were shown to play important roles in animal development. In this study, we cloned the p73 cDNA from zebrafish, using ovary RNA and methods of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Sequencing of four overlapping clones revealed a consensus sequence that contains a 1923-bp open reading frame (ORF) and flanking regions of 37-bp and 438-bp at the 5’- and 3’-ends, respectively. This ORF encodes a 640-amino-acid polypeptide which shares 95%, 71%, 71%, 70% and 32% homologies to p73 of Barbul barbus, Mus musculus, Homo sapiens, Cercopithecus aethiops and Mya arenaria, respectively. RT-PCR analysis revealed that zebrafish p73 was only expressed in restricted tissues such as skin, fin, brain, ovary and testis, in contrast to the more ubiquitous expression of zebrafish p53 and p63. During embryonic development, expression of p73 was detected at 3 hours post fertilization (hpf), and thereafter through 120 hpf. By whole mount in situ hybridization and immunohistochemical staining, we also found that the p73 was expressed in the olfactory bulb—like region and in specific cell types of the reproductive organs. The differences in spatial and temporal expression patterns among p73, p63 and p53 of zebrafish suggest that there are specific functional roles for the three genes.

目錄
頁次
中文摘要……………………………………………………………….1
英文摘要……………………………………………………………….2
背景介紹………………………………………………………………4
1. p53抑癌家族蛋白..……………………………………………...4
1-1. p53的基本結構……………………………………………...….4
1-2. p53的功能…………………………………………………...….5
1-3. p53家族成員--p73及p63……………………………………….7
1-4. p73和p63的結構……………………………………………….9
1-5. p73和p63的功能…………………………………………… ..11
2. 斑馬魚 (zebrafish)……………………………….……………….14
3. 實驗動機...………………………………………………………...17
實驗材料和方法……………………………………………………...18
1. 斑馬魚的飼養與繁殖...………………………….………….…….18
2. 萃取total RNA……………………………………………………18
3. RT-PCR…………………………………………………………....19
4. p73 cDNA選殖...……………………………………………..…...20
5. 3’RACE...………………………………………………...….…….25
6. 確定斑馬魚p73 cDNA序列並進行序列分析...………………....26
7. 斑馬魚各組織的RT-PCR分析...……………………………..…..27
8. 斑馬魚各胚胎期的RT-PCR分析...…………………………..…..27
9. 組織染色 (H&E)與免疫化學染色…………………………….....29
10. 全體原位雜交...……………………………………………….…..30
結果...………………………………………………………………....33
1. 斑馬魚p73 cDNA的選殖...………………………..………….….33
2. 斑馬魚p73與其它不同物種p73和p53的比較...………………..33
3. 斑馬魚p53家族成員在不同組織及胚胎期的表現...…………....34
4. p73在斑馬魚胚胎中的表現…………………………..…………..35
5. p73在卵巢的表現………………………………..………………..36
6. p73在睪丸的表現………………………………..………………..38
討論…………………………………………………………………...59
參考文獻……………………………………………………………...64
附錄…………………………………………………………………...64

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