2007-01-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/708582摘要：結合基因轉殖及複製動物技術，以中大型產乳經濟動物乳腺為生物反應器，從乳汁中生產具生物活性之高經濟價值醫療用生物製劑是台灣科學農業必然之發展方向。但產製基因轉殖中大型動物效率低、成本高，且常因質體架構不當或因蛋白質來源非同物種或非源自於乳腺，導致外源蛋白無法如預期分泌生產或因不當修飾作用而失活。以初級培養乳腺上皮細胞檢測外源基因表現可解決部分前述問題，但初級培養乳腺上皮細胞功能不穩定及無法長期繼代等缺點。以SV40 T-antigen可將細胞癌化而達到不朽化，可解決無法長期繼代缺點，但亦伴隨細胞轉形及喪失正常功能等缺點。表現端粒&#37238;於人類乳腺上皮細胞中亦能導致不朽化，其細胞形態及功能亦接近正常。本研究最終目的擬建立山羊不朽乳腺上皮細胞株，並作為測試以山羊乳腺為生物反應器時生產外源重組蛋白生物製劑之生產基因轉殖動物前的測試平台。本研究團隊於93年在農委會經費支持下已建立穩定表現端粒&#37238;之山羊乳腺上皮細胞株。這株細胞能表現乳腺上皮細胞特有標記，在Matrigel培養下亦能形成立體乳泡結構，具有乳腺上皮細胞應有特性。故本計畫擬於第一年深入研究其生化、遺傳、分化與泌乳能力等基本特性與提出專利申請，另外亦準備建立亞洲婦女穩定表現端粒&#37238;不朽化乳腺上皮細胞株。第二、三年則擬建立一個能在乳腺上皮細胞中可隨意調控之重組蛋白表現系統及純化系統，以克服當表現之重組蛋白會抑制乳腺功能時所遭遇之困難。最後，擬利用此不朽化乳腺上皮細胞生產純化人類的lactoferrin及對抗乳房炎的lysostaphin(溶菌&#37238;)，供作日後生產基因轉殖山羊的先期研究。成功建立此測試平台後，將能提供研究人員快速且正確的研究方向與實驗設計條件，節省資金及時間，提高基因轉殖經濟動物的產製能力。<br> Abstract: Production of recombinant protein biopharmaceuticals from domestic mammary gland, through a combination of transgenic and nuclear transfer technology, is a dominant system in scientific agriculture of Taiwan. Low efficiency, high costs and time-consuming are common problems in producing transgenic domestic mammalian; moreover, improper plasmid constructs, protein source from different species or other tissue, are more serious problems making protein lose biological activity. In order to solving some problems mentioned above, culture of the primary mammary epithelial cell is a feasible way, but cannot be cultured for long period. Using SV40 large T-antigen transformed cells to immortal state will accompany with carcinogenesis and lose normal cell cycle checkpoints control. Immortalization of cells with telomerase can get immortal cell line which is near normal cells both on morphology or function. In this project, we want to establish a telomerase- immortalized caprine mammary epithelial cell line serving as a platform of production of recombinant protein biopharmaceuticals in caprine mammary gland. Under funding support of Council of Agriculture, we had established a telomerase-immortalized caprine mammary epithelial cell line. Further, we had tested some characteristics of this cell line, like mammary epithelial cell specifically expressed cytokeratine 18 and formed alveolar structure on Matrigel matrix, showing this cell line still possess normal function of mammary epithelial cell. In this project, we will study the biochemistry, genetics, differential characteristics and milk-expressed ability on this cell line in the first year and to establish a telomerase-immortalized human epithelial cell line of Asiatic species. In the 2nd and 3rd years, we will set up an inducible expression system of recombinant protein in the immortal cell line and a protein purification system to overcome the problem that the recombinant protein may suppress normal function of mammary gland. In the last year, to be a pioneer of production of transgenic caprine, we will test the possibility of producing and purifying human lactoferrin and lysostaphin in this cell line. This platform may provide a rapid and correct way for researchers and may save money and time in promoting the production of transgenic animals.端粒&#37238不朽乳腺上皮細胞株生物反應器telomeraseimmortal mammary epithelial cell linebioreactor