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研究生:江宜勳
論文名稱:反股HBx在轉殖小鼠乳腺之作用
論文名稱(外文):Characterization of anti-sense HBx gene in the mammary gland of transgenic mice
指導教授:曾英傑曾英傑引用關係
學位類別:碩士
校院名稱:慈濟大學
系所名稱:分子生物及細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:反股HBx乳腺啟動子
外文關鍵詞:antisense HBx
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X基因為B型肝炎病毒(HBV)四個已鑑定基因之一,此基因可以編制(encode)出154胺基酸,且序列完整保留在所有哺乳類肝炎病毒中,例如:土撥鼠肝炎病毒woodchuck hepatitis virus (WHV)和松鼠肝炎病毒ground squirrel hepatitis virus (GSHV)。最近,我們實驗室發現了一個基因,其位置剛好落在B型肝炎病毒編譯序列的互補股(complementary strand) (在這裡,我們實驗室命名為Y 或是HBY基因)。藉由西方氏點墨法分析,在一位B 型肝炎病毒帶原者血清中,去偵測HBY接合蛋白,顯示出有一明顯的免疫反應。這暗示著HBY已經在自然界中表現。此篇論文的目的就是在以轉殖小鼠為研究模式,對這反向股HBx(antisense HBx),在轉殖小鼠的乳腺組織上進行探討。 本實驗之轉殖小鼠藉由乳腺特異性啟動子(WAP promoter)來表現HBY基因,基於此項設計,我們實驗室利用顯微注射法產生出一些親代(founders)小鼠,為偵測在乳腺上的功能是否受到HBY基因表現所影響。每間隔5天測量哺乳期轉殖小鼠的仔鼠體重,發現轉殖小鼠的仔鼠體重顯著輕於對照組野生型的仔鼠。基於這個發現,我們推測轉殖小鼠之母鼠的乳腺細胞,可能因為Y基因的誘導(induction) 而導致乳腺泌乳週期未結束前,就提早進入細胞凋亡期。因此,在泌乳週期中設定數個實驗觀察時間點,使用Hochest 33258來染乳腺組織切片,其結果顯示出在轉殖小鼠之母鼠的乳腺腔中出現了可能是細胞早熟而進入到細胞凋亡時期的細胞核。為確認細胞凋亡基因的活化作用,使用RT-PCR來偵測這些與細胞凋亡相關的基因表現,例如:TNF-R, Fas, Bax, BCL-xL, and C-myc,這結果也顯示出BCL-xL在轉殖小鼠之母鼠的乳腺泌乳期在第五天和第二十天明顯表現,然而,在對照組野生型的小鼠之母鼠卻沒有偵測到。 這些發現顯示出有可能HBY基因在轉殖小鼠乳腺組織上疑似有細胞凋亡現象。進一步研究則集中在肝癌病人的肝組織切片。
The X-gene is one of the four identified genes in the Hepatitis B Virus (HBV) genome. It encodes a polypeptide with 154 amino acids. The amino acid sequences of X protein are well conserved among mammalian hepadna virus, such as woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV). Recently our laboratory has found that a putative gene localized in the complementary strand of Hepatitis B Virus X (HBx) sequence (here, we named it as Y or HBY gene). By using Western blot analysis, antiserum from a HBV carrier showed significant immunity to recombinant HBY protein. This implicated that HBY gene has been expressed in the nature. The purpose of my thesis was to focus on characterization of the gene encoding antisense HBx in the mammary gland of mouse model. To this aim, we used the transgenic mice as a model. The transgenic mice expressed HBY gene driven by mammary gland specific promoter Whey Acidic Protein (WAP). Our laboratory has generated some founder mice. To detect whether the function of the mammary gland is influenced by HBY gene expression, the body weight of pups from transgenic mice was measured at 5 day intervals during suckling duration. The results showed that the body weight of the pups from transgenic females was significantly lighter than that of pups from normal ones. Based on this finding, we proposed that mammary cells might enter premature apoptosis due to HBY gene induction before end of lactation. Therefore, the tissue section of the mammary gland was examined by Hochest33258 stain at different intervals during lactation duration. The results indicated that mammary cells display a feature of premature apoptosis. To clarify the activation of apoptotic genes, the reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of TNF-R, Fas, Bax, BCL-xL, and C-myc apoptosis-related genes. The results indicated that Bcl-XL was detected in transgenic glands at day 5 and 20, however, undetectable in normal glands. These findings revealed that the mammary gland showed apoptosis-like effect by HBY gene expression. Further studies will focus on its impact on patient’s liver.
1 序論 6 1.1 B型肝炎病毒( Hepatitis B Virus, HBV) 6 1.2 B型肝炎病毒結構 7 1.2.1 開放譯讀序列及基因產物(open reading frames and their products) 7 1.2.2 調控要素(Cis-acting elements) 10 1.2.3 B型肝炎生活史 11 1.3 B型肝炎與肝癌( Hepatocellular carcinoma , HCC)行成之間的關係 12 1.4 HBx與HBV之間的關係 14 1.5 HBX gene 反股產物(antisense product)的發現及作用 15 1.6 基因轉殖技術之運用 17 1.7 本篇論文之目的 18 2 材料與方法 19 2.1 材料 19 2.1.1 DNA分析 19 2.1.1.1 自小鼠尾部粹取DNA 19 2.1.1.2 聚合酶連鎖反應PCR篩選基因轉殖小鼠 19 2.1.1.3 南方氏點墨法 (Southern blot) 20 2.1.1.3.1 利用限制酶 處理DNA 20 2.1.1.3.2 洋菜膠電泳分離DNA 20 2.1.1.3.3 轉漬DNA到尼龍膜上 20 2.1.1.3.4 探針的製備 21 2.1.1.3.5 雜交與呈色反應 21 2.1.2 RNA分析 22 2.1.2.1 粹取乳腺RNA 22 2.1.2.2 RT-PCR 22 2.1.2.3 北方氏點墨法 (Nouthern blot) 23 2.1.2.3.1 甲醛洋菜膠製作: 23 2.1.2.3.2 洋菜膠電泳分離RNA 24 2.1.2.3.3 轉漬RNA到尼龍膜上 24 2.1.2.3.4 探針的製備 24 2.1.2.3.5 雜交與呈色反應 24 2.1.3 蛋白質分析 25 2.1.3.1 培養細胞 25 2.1.3.2 MYIII發現 25 2.1.3.3 HBY接合蛋白構築 26 2.1.3.3.1 轉送(transfect)到細胞 26 2.1.3.3.2 細菌培養 26 2.1.3.4 粹取乳腺組織蛋白質 26 2.1.3.5 西方氏點墨法 26 2.1.3.5.1 蛋白質電泳膠片SDS-PAGE的製備 26 2.1.3.5.2 蛋白質電泳 27 2.1.3.6 蛋白質轉漬與Coomassie Blue染色 27 2.1.3.6.1 轉漬 27 2.1.3.6.2 Coomassie Blue染色 27 2.1.3.6.3 抗原偵測與呈色反應 27 2.1.3.7 免疫沉澱法 28 2.1.4 轉殖小鼠組織型態分析 28 2.1.4.1 TUNEL assay 28 2.1.4.2 石臘包埋切片 28 2.1.4.3 常規蘇木精與伊紅染色法HE stain (Hematoxylin and Eosin Staining) 29 2.1.4.3.1 蘇木精染色液Mayer's Hematoxylin 29 2.1.4.3.2 1% 伊紅酒精溶液 29 2.2 方法 29 2.2.1 轉殖基因的構築 29 2.2.2 基因轉殖小鼠篩選 30 2.2.3 DNA分析 30 2.2.3.1 自小鼠尾部粹取DNA 30 2.2.3.2 PCR篩選基因轉殖鼠 30 2.2.3.3 南方氏點墨法 32 2.2.3.3.1 探針的製備 32 2.2.3.3.2 雜交與顯影反應 32 2.2.4 RNA分析 33 2.2.4.1 粹取小鼠乳腺組織的總RNA 33 2.2.4.2 RT-PCR 33 2.2.4.3 北方氏點墨法 34 2.2.4.3.1 洋菜膠的製備 34 2.2.4.3.2 探針的製備 34 2.2.4.3.3 雜交與顯影反應 34 2.2.5 蛋白質分析 35 2.2.5.1 粹取HBY接合蛋白 35 2.2.5.1.1 培養細胞 35 2.2.5.1.2 在細胞株表現 35 2.2.5.1.3 在細菌表現 35 2.2.5.1.3.1 確認MYIII並轉送(transformation)到BL-21 35 2.2.5.1.3.2 誘導MYIII在細菌表達 36 2.2.5.2 粹取乳腺蛋白質 36 2.2.5.3 免疫沉澱法 36 2.2.5.4 西方氏點墨法 37 2.2.5.4.1 蛋白質電泳膠片SDS-PAGE的製備 37 2.2.5.4.2 蛋白質電泳 37 2.2.5.4.3 蛋白質轉漬與Coomassie Blue染色 37 2.2.5.4.4 免疫染色反應 38 2.2.5.5 轉殖小鼠乳腺組織型態觀察 38 2.2.5.5.1 WAP-HBY 轉殖小鼠哺育仔鼠狀況分析 38 2.2.5.5.2 石蠟組織切片 38 2.2.5.5.2.1 組織固定 38 2.2.5.5.2.2 組織脫水,清洗,浸潤 39 2.2.5.5.2.3 包埋 39 2.2.5.5.2.4 切片 39 2.2.5.5.2.5 脫蠟 39 2.2.5.5.2.6 染色 39 2.2.5.5.2.6.1 HE stain (Hematoxylin and Eosin Staining) 39 2.2.5.5.2.6.2 脫水 40 2.2.5.5.2.6.3 清洗 ( 透明 ) 40 2.2.5.5.2.6.4 封蓋 40 2.2.5.5.3 Tunnel assay 40 3 結果 41 3.1 以西方氏點墨法偵測B肝帶原者血清中是否含有HBY抗體 41 3.2 轉殖小鼠的產生 41 3.3 小鼠攜帶及表現轉殖基因之偵測 41 3.3.1 南方氏點墨法分析 41 3.3.2 WAP-HBY轉殖基因的表現偵測 42 3.3.2.1 利用RT-PCR分析 42 3.3.2.2 北方氏點墨法分析 43 3.3.2.3 西方氏點墨法 43 3.3.2.4 免疫沉澱法 43 3.4 WAP-HBY 轉殖小鼠生物效應 44 3.4.1.1 WAP-HBY 轉殖小鼠哺育仔鼠狀況分析 44 3.4.1.2 轉殖小鼠乳腺細胞凋亡分析 44 3.4.1.3 常規染色(HE stain)與DNA特異性染料(Hoechest 33258) 44 3.4.1.4 細胞凋亡試驗(TUNEL assay) 44 3.4.1.5 轉殖基因HBy對細胞凋亡基因的影響 45 4 討論 46 5 參考文獻 47 1
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