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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36326完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱有田(Yu-Ten Ju) | |
| dc.contributor.author | Meng-Wei Ke | en |
| dc.contributor.author | 柯孟韡 | zh_TW |
| dc.date.accessioned | 2021-06-13T07:57:08Z | - |
| dc.date.available | 2015-07-24 | |
| dc.date.copyright | 2005-07-30 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-24 | |
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Montagna, L. Susani, O. Barbieri, V. Pallini, P. Vezzoni, and R. Dulbecco. 2002. Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland. Proc. Natl. Acad. Sci. USA 99 (13) : 8660-8665. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36326 | - |
| dc.description.abstract | 自中大型產乳經濟動物乳腺產製醫療用重組生物製劑，是科學農業
未來的重點方向之ㄧ。但是，產製基因轉殖動物現今仍普遍存在低效 率、成本高及耗時長等問題。造成效率低的原因，主要有質體架構不當、 外源基因來自不同物種或組織，導致質體所表現的蛋白不具生物活性。 因此，若能先於體外培養之乳腺上皮細胞內，測試質體架構與表現的能 力，則可解決產製基因轉殖動物所遭遇的困難。惟體外初級培養之乳腺 上皮細胞有無法長期繼代培養之問題。利用大量表現致癌基因或γ 放射 線照射等方式，能夠造成細胞的不朽化，但是轉型後的細胞伴隨不正常 分化，甚至是癌化。穩定表現外源性端粒酶反轉錄酶，亦能誘導初級培 養之細胞成為不朽化，不論外形或分化功能上，皆與正常細胞較為相似。 本研究主要目的乃建立一株穩定表現外源性端粒酶之山羊乳腺上 皮細胞株 (CMEC)，做為產製基因轉殖動物質體架構之測試平台。初級 乳腺上皮細胞取自性成熟之雌性撒能山羊，利用cytokeratin 18 抗體進行 免疫螢光染色分析，證實CMEC 表現大量cytokeratin 18 蛋白質；同時， 當CMEC 長滿後，於接觸性誘導下，形成與活體乳腺發育中，乳腺泡內 腔類似的構造。進一步將CMEC 培養於MATRIGEL 上，更會被誘導形 成立體中空乳泡構造。經由上述定性分析，證實CMEC 保有乳腺上皮細 胞最基本而重要的功能。利用表現外源性人類端粒酶反轉錄酶於CMEC 中，經抗生素篩選後，已得到一株能夠穩定表現外源性端粒酶之山羊乳 腺上皮細胞株。 此細胞株之建立，不僅能做為產製基因轉殖動物質體架構的測試平 台，更能應用於研究乳腺發育調控、細胞衰老及癌症生成機制等領域。 於癌症應用上，已知Caveolin-1 基因扮演癌症生成的抑制角色 (如：乳 癌、結腸癌)；因此，為研究Caveolin-1 與癌症生成關係，本研究製備 Caveolin-1 胜肽多株抗體。此抗體以西方吸漬及免疫螢光染色法，證實 能夠辨識來自小鼠、山羊及人類等不同物種之內源性與外源性 Caveolin-1 蛋白質。此抗體進一步以免疫組織化學染色法，進行人類乳 癌、肺癌及直腸癌切片之測試比較，結果顯示，在乳癌及直腸癌切片試 驗中，與相同部位組織之正常切片做比較，此抗體可明顯區辨癌化與正 常切片中，Caveolin-1 表現的差異。證實此Caveolin-1 多株抗體，可應 用於人類乳癌及結腸癌之臨床診斷試驗。 結合穩定表現外源性端粒酶之山羊乳腺上皮細胞株與Caveolin-1 胜 肽多株抗體，將成為我們未來研究乳腺發育調控、基因轉殖動物質體架 構測試及細胞老化與癌化的重要利器。 | zh_TW |
| dc.description.abstract | Establishment of a stable telomerase-expressed caprine mammary epithelial cell line
Abstract Production of recombinant biopharmaceuticals from the mammary gland of domestic animals through transgenic nuclear transfer technology is a dominant system in scientific agriculture. Low efficiency, high costs and time-consuming, however, are common problems in producing transgenic domestic animals. Furthermore, improper plasmid constructs and gene source from different species or tissues are more serious problems making protein lose its biological activity. In order to solve the above problems, the culture of primary mammary epithelial cell for pioneer testing the expressed recombinant protein biological activity and characteristics is recommended, but the finite life span of cultured primary mammary epithelial cell is more concerned. SV40 large T-antigen transformed cells are also immortal but they accompany with carcinogenesis and lose of normal cellular functions. Stable expression of telomerase reverse transcriptase (TERT) in primary culture cells can induce cells immortalized and maintain cells with both normal cellular morphology and functions. The aim in this study is to establish a telomerase-immortalized caprine mammary epithelial cell line (CMEC) as a pioneer testing platform to examine the biological activity of interesting gene expressed in transgenic animal. In the beginning, the characteristics of the cultured CMEC were examined and verified it specifically expressed cytokeratin 18 and formed alveolar structure on MATRIGEL matrix, showing the CMEC still possess the normal function of mammary epithelial cell. Further, we transfected CMEC with hTERT and selected with proper antibiotics for establishing a stable hTERT-expressed caprine mammary epithelial cell line. As the immortal CMEC cell line is established, it can not only be used for mammary gland development study but also for cellular senescence and breast cancer research. Using mRNA subtractive hybridization and differential display techniques, caveolin-1 was seem to be lost or down-regulated in human mammary adenocarcinoma-derived cells comparing to normal mammary epithelial cell. For further researches, a rabbit polyclonal peptide antibody specially recognizing different species’ caveolin-1 protein was developed. This antibody could against 22-24 kDa caveolin-1 protein extracted from human epithelial cell line A431, CMEC, mice fibroblast cell line NIH3T3 and recombinant caveolin-1 transiently-expressed rat pituitary epithelial cell line GH3 by western blotting and immunostain techniques. This result had proved this antibody can be used for examining caveolin-1 protein expressed in mouse, rat, goat and human species. To explore whether this antibody can be applied for breast cancer diagnosis, we performed immunohistochemistry on breast tumor biopsy and showed significant difference in caveolin-1 expression between normal with tumor was observed. To the conclusion, once the immortal CMEC being established, we can study the development of mammary gland and the production of recombinant protein biopharmaceuticals. Further, combining with caveolin-1 antibody, the study of cellular senescence and signal transduction through caveolin-1 can also be achieved. | en |
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| dc.description.tableofcontents | 壹、摘要 ………………………………………………………………………………1
貳、前言 ………………………………………………………………………………3 參、文獻檢討 …………………………………………………………………………6 一、活體中乳腺發育與構造 ……………………………………………………6 (一) 乳腺構造 ………………………………………………………………6 (二) 影響活體乳腺發育因素 ………………………………………………7 1. 胜肽與固醇類內泌素 ………………………………………………7 2. 胞外基質 ……………………………………………………………8 二、體外培養下乳腺上皮細胞之發育 …………………………………………8 (一) 胞外基質對乳腺泡之發育調控 ………………………………………9 (二) 胜肽與固醇類內泌素對培養中乳腺細胞發育及功能之影響 ………9 1. 型態上的分化 ………………………………………………………10 2. 乳蛋白分泌調控 ……………………………………………………10 3. β-酪蛋白啟動子序列調控β-酪蛋白表現 ………………………11 三、乳腺上皮細胞的老化與不朽 ………………………………………………11 (一) 細胞衰老 ………………………………………………………………11 (二) 哺乳類動物細胞體外培養的衰老現象 ………………………………12 (三) 衰老細胞的生物性標記 ………………………………………………13 (四) 染色體端粒、端粒酶與細胞衰老之關係 ……………………………13 (五) 誘導乳腺上皮細胞不朽化的方法 ……………………………………14 (六) 經SV40 large T-antigen 轉型與端粒酶不朽化人類乳腺上皮細胞株之 比較 ……………………………………………………………………14 肆、材料與方法 ………………………………………………………………………15 一、山羊乳腺之取樣 ……………………………………………………………15 二、山羊乳腺上皮細胞之分離與初級培養 ……………………………………15 三、乳腺上皮細胞豐富化 ………………………………………………………16 四、細胞繼代培養及生長曲線之計算 …………………………………………17 五、細胞冷凍儲存 ………………………………………………………………18 六、細胞解凍活化 ………………………………………………………………18 七、萃取及純化大量質體DNA ………………………………………………18 八、細胞轉型感染 ………………………………………………………………19 九、細胞免疫染色 ………………………………………………………………20 十、抗生素篩選建立穩定表現外源性蛋白質之細胞株 ………………………21 十一、 不同胞外基質上之細胞培養 …………………………………………21 十二、 CMEC細胞total RNA之萃取 ………………………………………22 十三、 反轉錄 …………………………………………………………………22 十四、 聚合酶鏈鎖反應 ………………………………………………………23 十五、 Senescent associated β-Gal activity (SA β-Gal) stain …………………24 十六、 誘導CMEC 酪蛋白之表現 …………………………………………25 伍、結果 ………………………………………………………………………………26 一、山羊乳腺上皮細胞之分離 …………………………………………………26 二、乳腺上皮細胞之enrichment …………………………………………………26 三、山羊乳腺上皮細胞表現cytokeratin 18 蛋白質之檢測 …………………26 四、接觸性CMEC 培養促進類內腔構造形成 ……………………………………27 五、山羊乳腺上皮細胞體外培養之生長曲線 …………………………………27 六、山羊乳腺上皮細胞於不同生長代數之外觀變化 …………………………28 七、不同代數山羊乳腺上皮細胞SA β-Gal 活性測試 …………………………28 八、比較selection前後不同代數之山羊乳腺上皮細胞p16 gene 表現情 形………………………………………………………………………………29 九、乳泡狀構造之形成 …………………………………………………………29 十、hTERT 質體表現外源性端粒酶於山羊乳腺上皮細胞內 …………………30 十一、 抗生素篩選轉形感染後之山羊乳腺上皮細 …………………………30 十二、 山羊乳腺上皮細胞穩定表現外源性端粒酶 …………………………31 陸、討論 ………………………………………………………………………………32 一、 山羊乳腺上皮細胞之外觀與體外培養相關特性 ………………………32 二、 山羊乳腺上皮細胞體外培養之衰老現象 ………………………………33 三、 體外培養山羊乳腺上皮細胞p16 基因的表現變化 ……………………35 四、 表現外源性端粒酶於山羊乳腺上皮細胞 ………………………………35 五、 建立穩定表現外源性端粒酶之山羊乳腺上皮細胞株 …………………37 柒、結論 ………………………………………………………………………………38 捌、參考文獻 …………………………………………………………………………39 玖、英文摘要 …………………………………………………………………………44 壹拾、 附圖 …………………………………………………………………………45 圖一、山羊乳腺上皮細胞之分離 ……………………………………………………45 圖二、山羊乳腺上皮細胞之enrichment ……………………………………………46 圖三、Enrichment 前後，初級培養之山羊乳腺細胞表現cytokeratin 18 蛋白 質 ………………………………………………………………………………47 圖四、山羊乳腺上皮細胞形成類內腔構造 …………………………………………48 圖五、山羊乳腺上皮細胞體外培養之生長曲線 ……………………………………49 圖六、山羊乳腺上皮細胞於不同生長代數之外觀 …………………………………50 圖七、不同代數山羊乳腺上皮細胞SA β-Gal 活性測試 ……………………………51 圖八、比較selection 前後不同代數之山羊乳腺上皮細胞 p16 gene 表現情形 ……52 圖九、乳泡狀構造之形成 ……………………………………………………………53 圖十、pLPC-hTERT 表現外源性端粒酶於山羊乳腺上皮細胞 ……………………54 圖十一、pIRES2-hTERT 表現外緣性端粒酶於山羊乳腺上皮細胞 …………………55 圖十二、抗生素篩選轉形感染後之山羊乳腺上皮細胞 ……………………………56 圖十三、穩定表現外源性端粒酶之山羊乳腺上皮細胞株 …………………………57 附錄一、pLPC-hTERT mammary cell expressed plasmid ……………………………58 附錄二、pIRES2-hTERT mammary cell expressed plasmid …………………………59 附錄三、Development and Characterization of an Anti-caveolin-1 Peptide antiserum for Diagnosing Human Breast and Colon Cancer ……………………………60 附錄四、Bromocriptine Sensitizes Rat Pituitary GH3 Cells to Caveolin-1 Induced apoptosis ……………………………………………………………………87 | |
| dc.language.iso | zh-TW | |
| dc.subject | 山羊乳腺上皮細胞 | zh_TW |
| dc.subject | 端粒酶 | zh_TW |
| dc.subject | telomerase | en |
| dc.subject | caprine mammary epithelial cell | en |
| dc.title | 藉由端粒酶基因轉殖策略建立乳山羊不朽化乳腺上皮細胞株之
可行性 | zh_TW |
| dc.title | The feasibility of establishing immortal caprine mammary epithelial cell line by transfection of telomerase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 姜延年(Yan-Lien Jiang) | |
| dc.contributor.oralexamcommittee | 鄭登貴,王廷方 | |
| dc.subject.keyword | 端粒酶,山羊乳腺上皮細胞, | zh_TW |
| dc.subject.keyword | telomerase,caprine mammary epithelial cell, | en |
| dc.relation.page | 111 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-24 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 畜產學研究所 | zh_TW |
| 顯示於系所單位： | 動物科學技術學系 | |
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