2005-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/688364摘要：肺表面活性蛋白質係由肺產生，透過呼吸系統維持氧氣之吸收為其主要功能。在懷孕35週前胎兒表面活性蛋白質絕大部份都有表現量不足之缺陷，早產兒及具表面張力素遺傳缺陷之新生兒常發生嚴重之呼吸窘迫症狀。疏水性之肺表面張力素B及C在維持肺正常呼吸功能上扮演重要角色，肺表面張力素B及C基因發生突變將導致新生兒、兒童及成人肺功能失調及慢性間質性肺病變。肺表面活性蛋白質B和C一般係由牛或豬肺臟萃取而得，並常被用於治療該早產兒之呼吸窘迫症狀。目前每年之肺表面表面活性蛋白質製劑產量950公升，年產值超過九千萬美元。 乳腺在生理上屬較獨立之器官，由乳汁收集外源性蛋白質較容易且不具侵入性，因此被評估為量產醫藥蛋白質之最佳標的器官。相較於牛、山羊及綿羊，豬具有連續性發情週期、分娩多胎特性及較短之世代間距等之生殖特性，促使豬成為生產醫藥蛋白質之理想家畜，甚至可以機器搾乳。此外，山羊酪蛋白啟動子已被證實可組織專一性地大量表達外源性之醫藥蛋白質。因此，本研究擬以基因注入方式產製攜表面活性蛋白質B及C基因之轉基因小鼠及豬，藉由豬乳腺專一性表現及量產表面活性蛋白質B及C，作為醫療用之蛋白質。 以胚原核基因顯微注射法產<br> Abstract: Surfactants are substances that are produced naturally in the lungs and are essential to the lungs' ability to absorb oxygen and to maintain proper airflow through the respiratory system. Premature babies are born with a lack of natural surfactant in their lungs. Without surfactant, the air sacs in the lungs collapse and are unable to absorb sufficient oxygen resulting in respiratory distress syndrome (RDS). The hydrophobic surfactant proteins (SP), SP-B and SP-C, have important roles in surfactant function. The importance of these proteins in normal lung function is highlighted by the lung diseases associated with abnormalities in their expression. Mutations in the gene encoding SP-B result in severe, fatal neonatal lung disease, and mutations in the gene encoding SP-C are associated with chronic interstitial lung diseases in newborns, older children, and adults. So far, annual yield have reached 950 liters and output value reached US$ 90 million. The mammary gland is the best and isolated candidate organ of prodigious production system, capable of generating ample protein during lactation period, coupled with the relative ease of harvesting milk in a non-invasive manner. Moreover, several reproductive characteristics of the pig (continuous cyclicity, short generation internal, polytocious nature) make it an ideal choice of transgenic bioreactor for producing pharmaceutical products. Milking of pigs can even be mechanized. Caprine -casein promoter have been demonstrated to specifically express high levels of foreign protein in milk. Production of human SP-B and SP-C in a mammary gland bioreactor provides a potential method for generating sufficient amounts of SP-B and SP-C for addition to surfactant replacement mixtures. Pronuclear microinjection, which is still the only method routinely used to introduce foreign gene in the germ-line of mammalian species. The objects of this research proposal are to construct caprine -casein promoter with SP-B and SP-C cDNA, respectively, and coinject to pronucleus of fertilized eggs for generating SP-B and SP-C transgenic mice and pigs by pronuclear genes microinjection. In the first year, the qualitative and quantitative assay systems of transgenes expression will be established and performed as well as experiment of transgenic mice. The study in the second year will focus on the generation and screening of transgenic pigs harboring SP-B and SP-C by pronuclear microinjection. In the third year, germ-line transmission and specific expression of transgenes in mammary gland of transgenic founders will be confirmed. However, this project might be successfully done by our research team.肺表面活性蛋白質基因轉殖小鼠SurfactantTransgenic mice