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系統識別號 U0002-1708201011161000
中文論文名稱 Rrrm2在斑馬魚皮膚癌化過程中所扮演之角色
英文論文名稱 Ribonucleotide reductase m2(Rrm2)plays a role during zebrafish skin carcinogenesis
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 98
學期 2
出版年 99
研究生中文姓名 盧亭潔
研究生英文姓名 Ting-Jie Lu
電子信箱 judith0521@yahoo.com.tw
學號 697180346
學位類別 碩士
語文別 中文
口試日期 2010-07-16
論文頁數 66頁
口試委員 指導教授-陳曜鴻
委員-林明德
委員-周三芳
委員-王芸馨
中文關鍵字 斑馬魚  皮膚癌化 
英文關鍵字 zebrafish  skin carcinogenesis  rrm2 
學科別分類 學科別醫學與生命科學生物學
中文摘要 根據先前研究得知,在許多腫瘤細胞內shh訊號傳遞途徑會不斷被活化,進而造成細胞癌化而導致癌症的發生。其中基底細胞皮膚癌,basal cell carcinoma (BCC),就是因為 shh 訊號傳遞途徑發生問題所造成。我們於先前發表的paper中,發現當斑馬魚轉殖品系Tg(K18:shh-RFP)體內Shh過度表現時,會導致皮膚產生癌化現象,之後取其胚胎進行基因晶片(microarray)分析,發現魚體內rrm2基因表現量大幅上升(Chen et al.,2009),因此可知在shh過度表現情形下,會使rrm2被過度活化。
在建立另一斑馬魚轉殖品系Tg(K18:rrm2-RFP)過程中,發現部分注射過此質體之魚體於4天大時觀察到頭部、眼睛、胸鰭基部的皮膚發生變異,與野生品系相互比較之後發現此表現型疑似Tg(K18:shh-RFP),則初步懷疑shh與rrm2兩者間可能具關聯性;之後進行相關實驗觀察這些變異的皮膚組織是否與野生品系有不同之處。同時進行胚胎原位雜交反應(whole-mount in situ hybridization)確認rrm2在各個時期表現的訊號位置,結果發現訊號會表現在與細胞週期及細胞增生相關的位置上。
為更進一步研究rrm2與shh間的關聯性,將設計實驗推敲及證明兩基因間關係。取野生品系斑馬魚胚胎在發育約5.5小時時,分別浸泡不同濃度的cyclopamine用以抑制shh之功能,以及利用顯微注射(microinjection)技術將過量的K18-shh-RFP質體注射於胚胎內使魚體內shh過度表現,皆收取4天大之樣本,利用胚胎原位雜交反應觀察rrm2的訊號表現,是否會因為shh被抑制或過度活化而明顯降低或增加。
假使shh的基因表現與否會影響rrm2訊號表現,將試著搜尋rrm2上下游序列中是否具有Gli結合位,結果發現其上游有疑似Gli結合位的序列,則推測rrm2可能為shh所調控。過度表現的Shh會使rrm2表現增加,使細胞過度增生而可能導致皮膚表皮細胞癌化,本論文將探討rrm2與shh間關係且其在斑馬魚皮膚癌化過程中所扮演之角色。
英文摘要 According to previous studies that shh signaling pathway will continually be activated in many tumor cells and then cause cell carcinoma to lead into cancer. One of the basal cell skin cancer is basal cell carcinoma (BCC) which is caused by shh signaling pathway. In our previous studying, we found that when zebrafish transgenic lines Tg (K18: shh-RFP) Shh overexpress Shh in vivo, it will lead skin to have cancer phenomenon and then take the embryos for gene chip (microarray) analysis. We found rrm2 gene expression significantly increase in fish (Chen et al., 2009), so when shh is overexpressed , it will lead rrm2 into over-activation.
We generated the transgenic line Tg(K18:rrm2-RFP) that over-express rrm2 restrictively in the epidermis. We found some variation of the transgenic line by 4 days-post-fertilization(dpf), including that the mutation of head, eyes and the base skin of pectoral fin. Comparing with wild strain found that this phenotype is similar to the transgenic line Tg(K18:shh-RFP), then presume that may have the correlation between shh and rrm2.Through the experiments to test and verify the variety skin tissues that had different between Tg(K18:rrm2-RFP) and wild type(WT).To confirm the expression pattern of rrm2, we can use whole-mount in situ hybridization to display. Observing the expression pattern of rrm2,we found that rrm2 was expressed at the region of developing cell cycle and cell proliferation.
Futher,we designed a series of experiment to study the relevance of rrm2 and shh.We use cyclopamine and injected K18:shh:RFP plasmid to change shh expression in vivo.Silimarly,through observed the expression pattern of rrm2 demonstrate that shh was inhibited or overexpressed lead to rrm2 expressed addition or reduction.
If rrm2 was effected on shh,we tried to search the Gli binding site at rrm2 upstream or downstream sequences.Then,we found the silimar Gli binding site at rrm2 upstream sequences presumed that rrm2 would be regulated by shh.Shh was overexpressed that it increased rrm2 expression and caused skin carcinogenesis by inducing epidermal cells over-proliferation.In conclusion,we might discuss the connection between rrm2 and shh in this thesis.
論文目次 謝誌…………………………………………………………………Ι
中文摘要……………………………………………………………Ⅱ
英文摘要……………………………………………………………Ⅲ
目錄…………………………………………………………………Ⅳ
圖表目錄……………………………………………………………Ⅶ

第一章 前言………………………………………………………1
1.1 癌症的成因………………………………………………………1
1.2 皮膚的構造………………………………………………………3
1.3 皮膚癌的成因與種類……………………………………………3
1.4 Sonic hedgehog訊號之概述………………………………………4
1.5 Ribonucleotide reductase m2之功能與重要性…………………6
1.6 Sonic hedgehog與基底細胞皮膚癌之關係………………………9
1.7 Shh與rrm2之關係………………………………………………10
1.8 斑馬魚的好處……………………………………………………12
1.9 研究動機…………………………………………………………12
第二章 材料與方法…………………………………………14
2.1 斑馬魚(Danio rerio)飼養與胚胎收集…………………………14
2.2 大腸桿菌轉型法(transformation)………………………………15
2.3 聚合酶連鎖反應(polymerase chain reaction;PCR)……………15
2.4 小量質體DNA抽取……………………………………………16
2.5 質體建構(construction)………………………………………18
2.6 建立Tg(K18:rrm2-RFP)之品系……………………………19
2.7浸泡cyclopamine藥物與顯微注射質體K18-shh-RFP…………20
2.8原位雜交反應(Whole-mount in situ hybridization)……………21
2.9冷凍切片前處理及方法…………………………………………27
2.10 蘇木紫與伊紅染色(H&E stain)……………………………27
2.11 序列比對………………………………………………………28
2.12螢光顯微鏡、顯微照相系統及影像處理……………………28
2.13 rrm2 /shh-N double stain………………………………………28
2.14斑馬魚胚胎核酸之萃取………………………………………31
2.15反轉錄反應(reverse transcription-PCR)……………………31
第三章 結果……………………………………………………32
3.1 斑馬魚(Danio rerio)rrm2之分子結構與其他脊椎動物的演化關係…………………………………………………………………32
3.2 rrm2時間與空間上的分佈………………………………………33
3.3注射質體K18:rrm2:RFP與K18:shh:RFP 發現彼此表
現型(phenotype)極為相似…………………………………………35
3.4當shh過度表現或被抑制時,在眼睛周圍的rrm2訊號也明
顯隨之增減…………………………………………………………37
第四章 討論…………………………………………………………40
4.1 Shh pathway調控rrm2之表現…………………………………40
4.2 rrm2是否受到shh所調控?………………………………………41
4.3 抑制rrm2是否可以阻止癌化發生……………………………42
參考文獻……………………………………………………………44
圖表…………………………………………………………………51
附錄…………………………………………………………………63


圖表目錄
Fig.1(A)各物種間rrm2之同源序列比對…………………………52
Fig.1(B)各物種間rrm2之同源序列分析…………………………53
Fig.2內生性rrm2在早期斑馬魚胚胎上之表現……………………54
Fig.3內生性rrm2在晚期斑馬魚胚胎上之表現……………………55
Fig.4 K18-rrm2-RFP與K18-shh-RFP質體構築示意圖……………56
Fig.5顯微注射K18-rrm2-RFP與K18-shh-RFP質體表現型及組織切片染色圖……………………………………………………………57
Fig.6 rrm2/shh-N colocalization……………………………………58
Fig.7 shh overexpression & inhibition………………………………59
Fig.8 rrm2、shh與skin cell over proliferation間之關聯性…………60
Table.1斑馬魚rrm2與其他物種間之胺基酸相似度………………61
Table.2選殖及探針合成之引子序列………………………………62
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游宗翰 過度表現sonic hedgehog訊息導致斑馬魚皮膚癌化之分子機轉探討 淡江大學生科所碩士班碩士論文 96年6月
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