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研究生:曾媄慧
研究生(外文):Mei-Hui
論文名稱:研究人類p53promoter上對docetaxel之反應序列
論文名稱(外文):Investigation of the docetaxel response element at human p53 promoter
指導教授:許國堂
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學分子毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:68
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腫瘤抑制蛋白 p53在細胞受到損害的時候,扮演了保護細胞的重要角色。
在有外在的壓力下會藉由許多的機制去調控活化與誘導 p53基因的轉錄,但是這些機制與調控的路徑至今依舊不清楚。從先前的實驗結果中,我們發現了Docetaxel (Doc)會誘導 p53基因的轉錄活性,為了找到Doc在 p53 promoter上的反應序列,更進一步在 p53 promoter上設計了許多不同部位的缺失進行研究。由於 p53 promoter上並沒有TATA或是 GC box的相似序列,在判斷 p53 promoter的活性上具有一定的困難度,因此,我們利用Luciferase的方法去分析 p53 promoter本身的活性區域。本論文中,轉錄起始點開始往下游的約 120個核甘酸序列對於 p53 promoter本身活性也具有重要的影響。同時,在我們將構築的p53 promoter部分缺失的序列做 Doc的加藥處理,相較於控制組可以發現在 p53 promoter缺失序列中,p-200是所構築的序列中可以受到 Doc影響促使 p53 promoter活性上升的最小區域片段。已知有部分轉錄因子會反應在 p53 promoter上的序列,有一段約21 bp長的序列可以受到像UV或是其他化療藥物的刺激,進而促使 p53 promoter的活性上升。推測 Doc可能也是藉由在此區域影響 p53 promoter活性,因此 p-200序列中的 PE 21區域設計突變序列分別命名為M1、M2、M3、M4。將構築完成的PE 21突變序列短暫轉染至A54ADB2茩M後加藥處理,利用報導基因的方法去偵測突變序列對於 Doc藥物刺激之活性反應,發現 M4的序列對於藥物的刺激無活性上升的反應。將 M4的序列比對資料後,發現與受到 5-FU化療藥物刺激在 p53 promoter反應序列相重疊。以5-FU的藥物刺激A549細胞,發現會誘導內生性 p53 RNA與蛋白表現量上升;短暫轉染 p53 promoter序列與 PE 21突變序列在 5-FU的刺激下,除了 M4之外其他的序列依舊可以受到 5-FU刺激。因此,推測在 p53 promoter 上,Doc與 5-FU可能擁有相同的反應序列。
The p53 tumor suppressor protein plays a central role in the cellular defence against agents which cause genetic damage. The induction and activation of p53 upon stresses have been shown at post-transcription level by multiple mechanisms, while the regulatory role of p53 gene transcription is still poorly understood. From our laboratory previous data, we found that Docetaxel ( Doc) induces p53 transcription activity. To find Doc response element at p53 promoter, we created several deletion constructs of p53 promoter for this study, Because there is no similar sequences to TATA box or GC box, so we could not distinguish the basal activity of p53 promoter. To solve this problem, we used luciferase reporter assay to analysis basal activity of p53 promoter. In this thesis we confirmed that a 120 bp sequences from downstream of transcription start site is important for p53 promoter basal activity. In the same, we also transfection p53 promoter deletion constructs into lung cancer lines with Doc treatment, tha results showed, p-200 is the shortest fragment of p53 promoter, which can be stimulated by Doc and expressed basal activity. It has been reported that a 21- bp (PE 21) element can up regulate p53 promoter activity by treated with chemical /medicine or UV. Therefore, we suspected the regulation of Doc on p53 promoter is also through this PE 21 element. To identify the truly response element of Doc, we constructs four different services deletion constructs on PE 21 (M1-M4) sequences. The data revealed that, only M4 has no response to Doc stimulation. In comparison with previous studies, we found that 5-FU not only up regulates p53 RNA and protein, but also acts with similar sequences of Doc response element. In further experiment we found that all the constructs can be regulated by 5-FU except M4. Considered all the results, we hypothesized Doc probably has same response element of 5-FU induction.
壹、
中文摘要
1
貳、
英文摘要
2
參、
縮寫表
3
肆、
緒論
4
一、肺癌
4
二、化療藥物
5
1.Docetaxel〈taxotere〉
2. Doxorubicin 〈Adriamycin: ADR〉
3.5-fluorouracil〈5-FU〉
5
5
6
三、p53
6
1. p53基因功能
6
2. p53 promoter背景特性
7
伍、
研究動機
9
陸、
實驗材料與設備
10
一、實驗材料
10
1.藥品及試劑
10
2.酵素
10
3. Kit
10
4.其他
11
二、實驗設備
11
柒、
實驗方法
12
一、p53 promoter質體之構築
12
1.p53 Promoter各個長度片段之製備
12
2.洋菜凝膠(agarose gel)之電泳分析
14
3.由agarose gel中回收DNA片段(DNA Elution)
15
4.yT&A質體接合作用(TA ligation)
16
II
5.轉型作用(Transformation) 17
6.Colony Polymerase Chain Reaction(Colony PCR) 17
7.微量質體DNA 抽取(Miniprep) 18
8.中量質體DNA 抽取(Midiprep) 19
9.限制酵素反應(Restriction Enzyme digestion) 20
10.質體與insert DNA 片段的接合反應(ligation) 20
11.自動定序反應的製備(autosequence) 20
二、細胞培養(Cell culture) 21
1.解凍細胞株(frozen cells thawing) 21
2.細胞株培養(cell culture) 22
3.細胞繼代培養(passage)及種細胞 22
4.回凍細胞(frozen cell) 22
三、細胞轉染作用(Transfection) 23
1.TransFastTM Transfection Reagent 23
2.TurboFectTM in vitrro Transfection Reagent 24
四、Luciferase assay system 24
1.收細胞 24
2.s-galactosidase reporter gene assay 25
3.Luciferase reporter gene assay 25
五、西方點墨法 (Western blotting) 25
1.樣品製備 25
2.蛋白濃度定量分析 26
3.SDS-聚丙烯醯胺板膠之製備與操作 26
4.抗體作用及偵測 27
六、細胞中 RNA 萃取 27
七、cDNA 的合成與鏈聚合酵素連鎖反應 28
1.以DNaseⅠ處理細胞RNA 28
2.RNA 之反轉錄作用 29
3.RT-PCR 29
捌、 實驗結果 31
III
一、Docetaxel 對 p53 RNA 與蛋白之誘導 31
二、p53 promoter 的活性與Docetaxel 的濃度關係 31
三、Docetaxel 對 p53 promoter 有持續誘導的效果 32
四、比較 p53 promoter 上的序列原本的活性
五、比較 p53 promoter 上部分缺失對於 Docetaxel 的反應
33
34
六、PE 21 突變序列影響對 Docetaxel 的反應 35
七、PE 21 突變序列對於 p53 promoter 本身活性影響 36
八、在5-FU 刺激下p53 RNA 與蛋白質的表現量 36
九、5-FU 可以誘導 p53 promoter 的活性 37
十、5-FU 與Docetaxel 作用在 p53 promoter 上的反應序列可能相同 37
玖、 討論 39
拾、 圖表說明 43
圖一、細胞之 p53 RNA 與蛋白質受到 Docetaxel 之誘導 43
圖二、p53 promoter 的活性與Docetaxel 的濃度成正比反應 44
圖二、Docetaxel 對 p53 promoter 有持續誘導的效果 45
圖三、比較 p53 promoter 上的序列原本的活性 46
圖四、比較 p53 promoter 上部分缺失對於 Docetaxel 的反應 47
圖五、PE 21 突變序列影響對 Docetaxel 的反應 48
圖六、PE 21 突變序列對於 p53 promoter 本身的影響 49
圖七、在5-FU 刺激下p53 RNA 與蛋白質的表現量 50
圖八、5-FU 可以誘導 p53 promoter 的活性 51
圖九、5-FU 與Docetaxel 作用在 p53 promoter 上的反應序列可能相

52
表一、p-200 序列為 p53 promoter 部分缺失序列中最具代表性 53
表二、M4 突變序列不受到 Docetaxel 影響 54
表三、5-FU 與 Docetaxel 對於 M4 突變序列無影響能力 55
拾壹 附表及附圖 56
拾貳、 參考文獻 61
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