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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳義雄 | |
| dc.contributor.author | Tsai- Fu Hsieh | en |
| dc.contributor.author | 謝才富 | zh_TW |
| dc.date.accessioned | 2021-06-13T04:48:07Z | - |
| dc.date.available | 2011-08-08 | |
| dc.date.copyright | 2011-08-08 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-27 | |
| dc.identifier.citation | 1. Clark JH, markaverich BM. Actions of ovarian steroid hormones. The Physiology of Reproduction Raven, N. Y.; 1988. p. 675-711.
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Proc Natl Acad Sci U S A 1983;80(11):3334-8. 67. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422(6927):37-44. 68. Phonphok Y, Rosenthal KS. Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines. FEBS Lett 1991;281(1-2):188-90. 69. Block K, Kardana A, Igarashi P, Taylor HS. In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing mullerian system. FASEB J 2000;14(9):1101-8. 70. Li S, Hansman R, Newbold R, Davis B, McLachlan JA, Barrett JC. Neonatal diethylstilbestrol exposure induces persistent elevation of c-fos expression and hypomethylation in its exon-4 in mouse uterus. Mol Carcinog 2003;38(2):78-84. 71. Li S, Ma L, Chiang T. Promoter CpG methylation of Hox-a10 and Hox-a11 in mouse uterus not altered upon neonatal diethylstilbestrol exposure. Mol Carcinog 2001;32(4):213-9. 72. Yoshida A, Newbold RR, Dixon D. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33573 | - |
| dc.description.abstract | 利用二維電泳比較由人工合成己烯雌酚 (DES, diethylstilbesterol) 誘導幼鼠所產生之子宮液與正常成鼠於動情週期所產生之子宮液兩者蛋白質的成分，發現一個分子量為 14 kDa 、等電點為 4.6 的蛋白於成鼠表現，但於 DES 誘導的幼鼠沒有表現。由 MALDI-Q-TOF 質譜與 N端分析，證實此一蛋白質為 proline-rich acidic protein (PRAP)。
檢視成鼠性腺，Prap mRNA僅於子宮內膜細胞表現。於動情週期中， Prap mRNA從動情前期 (proestrus) 開始表現，於動情期 (estrus) 表現明顯增加，至早期動情後期 (metestrus I) 達到最高峰，於後期動情後期 (metestrus II) 快速遞減，最後於動情間期 (diestrus) 則幾乎沒有表現。 對未成熟的小鼠施予雌性素 (17 β-estradiol) (0.01- 10 μg/g body weight) 三天後，皆於子宮中檢視到Prap mRNA 與PRAP蛋白質的表現，但表現量隨著雌性素劑量增多而減少。單獨施打黃體素 (progesterone) 並不引發Prap 的表現，共同施打雌性素和黃體素，並沒有抑制雌性素的刺激。進一步證實Prap 受雌性素的刺激和雌性素α亞型有關。三週大幼鼠施予連續三天DES (0.1 μg/g body weight) 誘導的子宮液沒有發現PRAP。若先施打DES於未成熟小鼠，待其年齡到達成熟週數，Prap 基因在子宮的表現，相對於正常成鼠也顯著減少。 重組PRAP 蛋白外加於培養液會內吞至人類子宮內膜腺癌細胞 (Ishikawa cells)，雌性素會提高人類子宮內膜腺癌細胞的鹼性磷酸酶酵素活性及其反應基因，單獨外加重組PRAP 蛋白並無影響，若同時加入雌性素和PRAP， PRAP 加強雌性素所刺激的鹼性磷酸酶酵素活性及其反應基因。 | zh_TW |
| dc.description.abstract | Mice were used as experimental animals to study the functional role of uterine PRAP and how its expression is regulated by ovarian steroids. Soluble proteins in the uterine luminal fluid (ULF) of estrous females were resolved by 2-D gel electrophoresis. The protein spot corresponding to PRAP on the gel slab was identified by proteomic analysis. However, PRAP was absent in the ULF of DES- treated immature mice. Among the sexual glands of adults, both PRAP and its transcript were exclusively expressed in the endometrial epithelium of estrous females. Uterine Prap expression changed during the estrous cycle, in which the transcript levels progressively increased from proestrus via estrus to early metestrus, after which they rapidly declined via late metestrus to a very low level in diestrus. Daily injection of estradiol (E2) at a dose of 0.1 μg•g-1•day-1 to immature females for 3 consecutive days markedly stimulated uterine Prap transcription, whereas transcription was not initiated in the animals that received an equal dose of diethylstilbestrol dipropionate (DES). Relative to the normal estrus female, prepubertal DES exposure decreases remarkedly in the uterine Prap expression when the animals became sexually matured. The E2-stimulated Prap transcription involved the α-type estrogen receptor; at higher E2 doses, a lower level of uterine Prap mRNA and a lower amount of PRAP protein in ULF were detected. Using the Ishikawa cell line as a surrogate model of endometrial epithelium, we demonstrated that exogenous PRAP in the milieu rapidly internalizes into the cells to enhance endometrial estrogen responsiveness. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T04:48:07Z (GMT). No. of bitstreams: 1 ntu-100-D93b46011-1.pdf: 4870583 bytes, checksum: ffab0b62751747373062b0eed0b5da9b (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 i 謝辭 ii 中文摘要 iii Abstract iv 第一章 概論 1 生殖系統 1 雌性生殖 2 子宮液生成及相關重要研究 4 雌性素受體 5 DES之介紹 6 人類子宮內膜腺癌細胞株 6 Proline-rich acidic protein (PRAP) 簡介與相關研究 7 主要研究方向與實驗背景 8 第二章 材料與方法 9 實驗材料 9 實驗方法 9 一、小白鼠子宮組織及子宮液的樣品製備 9 1.1 類固醇激素的處理 9 1.2 動情週期各時期之篩選 9 1.3 類雌性素作用探討 10 二、蛋白質的分析方法 10 2.1 蛋白質定量 10 2.2 二維電泳 (Two-dimension electrophoresis) 11 2.3 SDS-聚丙烯醯胺膠體電泳 13 2.4 蛋白質染色分析 15 2.5 MALDI –Q – TOF 蛋白質樣品處理 17 2.6 N- 端定序 18 三、分子生物學相關研究 19 3.1 Total RNA 抽取 19 3.2 RNA品質的檢定： 20 3.3 北方點墨法 (Northern blotting) 21 3.4 反轉錄-聚合酶連鎖反應 (RT-PCR) 25 3.5 即時聚合酶鏈反應 (Real-time PCR) 26 3.6 雷射顯微擷取儀 (Laser capture microdissection) 27 3.7 Computational analysis 演化樹分析 28 四、以基因重組技術表現 PRAP 28 4.1 聚合酶連鎖反應 28 4.2 DNA 片段溶離 29 4.3 限制酶反應 29 4.4 DNA 接合反應 30 4.5 質體轉型 (Transformation) 30 4.6 表現載體之誘導 31 4.7 純化PRAP表現蛋白 31 五、蛋白質之偵測 32 5.1製備抗體 32 5.2 西方墨點法 33 5.3 免疫組織化學法 34 六、人類子宮內膜腺癌細胞株細胞實驗 35 6.1 細胞培養 35 6.2 鹼性磷酸酶酵素活性測定 36 6.3 雌性素活性反應基因偵測 36 6.4 PRAP 蛋白標記 37 6.5 PRAP 內吞作用 38 七、統計分析 39 第三章 結果 40 一、比較成鼠和DES刺激幼鼠之子宮液蛋白 40 1.1 子宮液蛋白的解析 40 1.2 基因重組技術表現PRAP及抗體之製備 40 1.3 子宮分泌PRAP之偵測 41 1.4 Prap 之序列分析 41 二、證明Prap於子宮內膜細胞表現 42 2.1 Prap基因在小鼠性器官中的組織分布 42 2.2 Prap基因在子宮的表現位置 42 三、 Prap於子宮動情週期中的表現 43 3.1 Prap於動情週期之變化 43 3.2 Prap於各動情週期之表現位置 43 四、 評估卵巢類固醇調控子宮內 Prap 的表現 43 4.1 Prap 與類固醇激素的關係 43 4.2 Prap在類固醇激素刺激之子宮表現位置 44 4.3 雌性素對 Prap 表現的影響 44 4.4 Prap 表現與雌性素受體之探討 45 五、尋找 PRAP的生殖功能 45 5.1 PRAP 的內吞實驗 45 5.2 外加 PRAP 會強化E2刺激Ishikawa cells所提升的鹼性磷酸酶酵素活性 46 5.3 PRAP 對於雌性素反應基因的之影響 46 六、己烯雌酚刺激幼鼠會影響成鼠子宮內 Prap 的表現 46 第四章 實驗討論 48 一、卵巢類固醇激素與子宮中 Prap 表現的關係 48 二、雌性素受體與Prap 表現的關聯性 49 三、PRAP 的內吞作用意義 49 四、己烯雌酚與小白鼠 Prap 之關聯性 50 圖次 52 附圖 82 附表 87 參考文獻 88 附錄 94 | |
| dc.language.iso | zh-TW | |
| dc.subject | 鹼性磷酸酶 | zh_TW |
| dc.subject | 己烯雌酚 | zh_TW |
| dc.subject | Prap | zh_TW |
| dc.subject | 子宮內膜腺癌細胞 | zh_TW |
| dc.subject | 酵素活性 | zh_TW |
| dc.subject | 內吞作用 | zh_TW |
| dc.subject | α-type estrogen receptor | en |
| dc.subject | diethylstilbestrol | en |
| dc.subject | uterine luminal fluid | en |
| dc.subject | Ishikawa cell line | en |
| dc.subject | estrogen responsiveness | en |
| dc.title | 一個多脯胺酸酸性蛋白在子宮內膜細胞的轉錄調控和加強該細胞對雌性素反應的能力 | zh_TW |
| dc.title | The estrogen-dependent transcription of an endometrial proline-rich acidic protein PRAP and its ability to enhance endometrial estrogen responsiveness | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 潘榮隆,梁博煌,李明亭,黃彥華,李勝祥,林翰佳 | |
| dc.subject.keyword | 己烯雌酚,Prap,子宮內膜腺癌細胞,內吞作用,鹼性磷酸酶,酵素活性, | zh_TW |
| dc.subject.keyword | uterine luminal fluid,diethylstilbestrol,α-type estrogen receptor,estrogen responsiveness,Ishikawa cell line, | en |
| dc.relation.page | 94 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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