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研究生:王俊仁
研究生(外文):Chun Jen Wang
論文名稱:利用四環素開啟表現系統探討ATM基因在不同組織的特異性
論文名稱(外文):Exploring Tissue Specificity of ATM by using the Tet-on system
指導教授:沈志陽沈志陽引用關係徐邦達徐邦達引用關係
指導教授(外文):C.Y.Shen, Ph.D.B.D.Hsu,Ph.D.
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
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:86
中文關鍵詞:乳癌離子放射線細胞週期ATM基因
外文關鍵詞:Ataxia-Telangiectasiaataxia-telangiectasia mutatedATMDoxycyclineantisense RNAbreast Carcinomap53chk2p95/NBS1
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中文摘要:
Ataxia-Telangiectasia(A-T)是人類的隱性遺傳疾病,是由於ATM基因發生突變所引起,ATM所扮演的角色為辨認由離子放射線(IR)所引起的DNA傷害,和調控數個細胞週期checkpoints的活化,進一步調節細胞週期的進行。從流行病學的研究中了解A-T異分子(heterozygotes)的人,有增加上皮細胞癌症發生的危險率,特別在乳癌。所以本研究想要了解ATM異常蛋白減少時,其下游基因的反應,在不同組織是否有差異性存在,以了解為何A-T異分子的人士以乳癌的發生,為主要的疾病。本研究利用Tet-on 表現系統作為表現方式,其表現特徵會因輔因子Doxycycline的增加而增加表現量。藉由此系統產生不同量的ATM antisense mRNA,以抑制ATM mRNA,進一步減少ATM蛋白的產生,然後再以免疫墨點法(Immunoblooting)來分析ATM下游基因(p53,chk2,p95/NBS1)的反應。從實驗的結果得知ATM蛋白在細胞中的含量會因doxycycline增加而減少,而下游基因在MCF-7(Breast Carcinoma),Hep G2(Human Hepatocellular Carcino- ma),293T(Human Embryonic Kidney Cell) 和Jurkat(Human acute T-cell Leukemia) 四種組織細胞中的表現並沒有因ATM蛋白表現的減少而有明顯的減少,只在完全沒有ATM存在時才會不表現。所以就結果來看,本實驗利用tet-on表現系統來表現antisense mRNA,以抑制ATM在細胞中的含量是可行的,而當ATM的表現量減少時,其下游所調控的蛋白並不會有明顯的減少,這或許是說明,雖然只剩下少量的ATM蛋白,但仍然有正常的功能,只是會比未表現antisense mRNA的情況下來的弱。
Ataxia-telangiectasia (A-T) is a recessive genetic disease that progressively and degenerately affects a startling variety of body systems. Several epidemiological studies have found that patients with A-T heterozygotes tend to have higher risk to develop epithelial carcinoma, especially breast carcinomas. Although tumorigenic mechanisms to explainthe occurance of tissue specificity is not clear, the gene responsible for A-T (ataxia-telangiectasia mutated;ATM) has been identified recently, and it is believed that ATM plays a key role In tumor suppression this disease.
In this study, we aimed at investigating how ATM down-stream ptoteins react againt ioizingradition when the ATM protein were down-regulated,and we focus on the differences among cells of tissue origins.
The Tet-on gene expression system, using Doxycycline as a cofactor to control the gene expression, was used to generate an ATM antisense mRNA to suppress the expression of ATM protein. Immunoblotting was used to monitor possible functional alteration of ATM down-stream genes (p53, chk2, p95/NBS1).
Our data shows that the ATM protein was successfully down regulated by adding increasing concentration of Doxycyclin in Tet-on gene expression system. however, no significantly functional differences of ATM down-stream genes was found in MCF-7 (Breast Carcinoma), HepG2 (Human Hepatocellular carcinoma), 293T (Human embryonic kidney cell) and Jurkat (human acute T-cell Leukemia). Only when the ATM protein was totally absent, the function of ATM down-stream protein was abolished.
In conclusion, the outcome of using Tet-on system expressing antisense mRNA to inhibit the expression of ATM within the cells is good. When the ATM expression was down regulated, the expression of the downstream regulated proteins did not decrease significantly. This phenomenon could be explained as even low amount of ATM was expressed, but all function normally, therefore, the effect of down regulated ATM downstream proteins is slightly weaker than those not expressing antisense mRNA
目錄
中文摘要                1
英文摘要                2
前  言                3
材料與方法               10
結  果                27
討  論                32
圖  表                37
參考文獻               82
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