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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 朱有田(Yu-Ten Ju),姜延年(Yan-Nian Jiang) | |
dc.contributor.author | Chia-En Huang | en |
dc.contributor.author | 黃佳恩 | zh_TW |
dc.date.accessioned | 2021-06-15T06:10:46Z | - |
dc.date.available | 2011-08-22 | |
dc.date.copyright | 2011-08-22 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-18 | |
dc.identifier.citation | Allar, M. A. and T. L. Wood. 2004. Expression of the insulin-like growth factor binding proteins during postnatal development of the murine mammary gland. Endocrinology 145: 2467-2477.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47652 | - |
dc.description.abstract | 第一型類胰島素生長因子(type 1 insulin-like growth factor, IGF-1)是一具有多功能之生長因子,包括與細胞生長、細胞分化調控及抑制凋亡等功能。IGF-1主要藉由其受器(insulin-like growth factor receptor, IGF-1R)活化來調控細胞功能。IGF-1R與胰島素受器構造相似,皆由四聚體構造所組成,胞質內脂質蛋白質片段具有酪胺酸激酶活性區域。IGF-1之活性受許多因子調控,其包含了六種具有高親和力之結合蛋白(type 1 insulin-like growth factor binding protein 1 to 6, IGFBP-1 to -6)以及可與IGFBPs作用之分子。於小鼠早期發身期時,IGF-1表現於乳腺組織中非上皮細胞部分,並於發身期中乳管發育時,IGF-1會表現於終端乳腺芽(terminal end bud, TEB)之上皮細胞及乳腺周圍基質中;當懷孕晚期時,IGF-1則會表現於乳泡(alveolar)及乳管上皮細胞中。上述於特定時期對於乳腺中特定部位IGF-1表現之調控,推測其應於小鼠乳腺發育中具有重要之功能,但對於山羊此一經濟動物之功能仍尚未釐清。
從山羊耳朵組織中選殖之IGF-1(gIGF-1)cDNA,其長度約為465個核苷酸。gIGF-1 cDNA與不同物種間之胺基酸序列相似度為70%至90%。繼之將gIGF-1 cDNA架接於帶有人類巨細胞病毒啟動子(cytomegalovirus promotor, CMV promotor)之後,構築可將gIGF-1於哺乳動物細胞中表現之質體,經由轉染將gIGF-1暫時表現於不朽化山羊乳腺上皮細胞株(immortalized caprine mammary epithelial cell line, CMC)中,再進一步利用細胞免疫螢光染色觀察gIGF-1於細胞株中是否能表現。結果確定gIGF-1能表現於細胞中,繼之檢測細胞株是否具有能力可將重組gIGF-1蛋白質分泌至細胞外。另外為了解決在CMC中細胞株短暫表現gIGF-1轉型效率不一而導致細胞分泌重組gIGF-1蛋白質效率降低之疑慮,本研究亦用大腸桿菌表現重組蛋白質系統(pET/ E. coli expression system)產製成熟態、訊息胜肽保留態兩種重組gIGF-1蛋白質,可藉由外源性添加一定濃度之gIGF-1來探討其與CMC細胞株生長之關係。本重組質體已架構完成,未來gIGF-1之重組蛋白質,於大腸桿菌中可經由外源性添加異丙基–β–d–1–硫代半乳糖苷(isopropyl β-D thiogalactopyranoside, IPTG)此一藥物,誘導重組gIGF-1之蛋白質產生。經純化之重組gIGF-1蛋白質,日後可於CMC細胞株中進行gIGF-1功能性探討。 綜合上述與依據細胞免疫螢光染色之結果得知選殖gIGF-1 cDNA於所構築之載體可成功表現於CMC細胞株中,而重組gIGF-1蛋白質是否會分泌至細胞外仍需進一步確認。 | zh_TW |
dc.description.abstract | Type 1 insulin-like growth factor (IGF-1), is a pluripotent growth factor and has been implicated in all phases of cell cycle dynamics, including promoting cell proliferation, differentiation and inhibiting cell death. IGF-1 applies its actions primarily through the type 1 insulin-like growth factor receptor (IGF-1R), which possesses a heterotetrameric structure with internal tyrosine kinase activity and structural homology to the insulin receptor. The bioactivity of IGF-1 is controlled by a complex system which consists of six high affinity binding proteins (IGFBP-1 to -6) and other IGFBP-interacting molecules. In mouse, IGF-1 expressed in the mammary gland nonepithelial portion in early puberty, and expressed in the epithelial cells of the terminal end buds as well as the stromal compartment during the pubertal ductal growth, and also will express in the alveolar and ductal epithelium during late pregnancy age. The spatially and temporally restricted expression of IGF-1 in the epithelium proposed that it may have the important function in the development of mouse mammary gland, but in one of the economic animals, goat, is not clear yet.
A goat IGF-1 (gIGF-1) cDNA (465 base pairs) was cloned from ear tissue. The similarity of amino acid sequences between species is from 70% to 90%. The gIGF-1 cDNA was subcloned into a mammalian expression vector and derived by a human cytomegalovirus (CMV) promoter. The gIGF-1 act as a transient expression in an immortalized caprine mammary epithelial cell line (CMC). The localization of gIGF-1 in CMC was observed by immunocytochemistry approach. The expression pattern inferred to drive endoplasmic reticulum-golgi (ER-golgi) pathway that may go through the auto-paracrine by secreting gIGF-1 out of the CMC cell. In order to overcome the question of unpredictable amount of transient expression of gIGF-1 in CMC, the pET/E. coli expression system will apply to express the mature, signal peptide fused, and gas7 gene fused recombinant gIGF-1 proteins. With all the the constructions mentioned above, the recombinant gIGF-1 protein will be induced expression by the isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction. The scaled-up recombinant gIGF-1 protein purified from the pET system will be used to investigate the function of gIGF-1 in CMC in the near future. To sum up, the immuno-staining result indicated that the constructions mentioned above could express in CMC, and according to the pattern which expressed in the cell, it is inferred that the recombinant gIGF-1 in cells transport follows the endoplasmic reticulum-golgi (ER-golgi) pathway. The confirmation whether the recombinant gIGF-1 can secrete out of cells needs further evidence. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:10:46Z (GMT). No. of bitstreams: 1 ntu-100-R97626015-1.pdf: 2315544 bytes, checksum: 7b794f69152877eaf8d7682c633226f4 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 中文摘要 I
英文摘要 III 目錄 V 圖次 VIII 表次 IX 前言 1 文獻檢討 2 一、乳腺組織之發展 2 (一)胚胎時期之乳腺生成 2 (二)出生後之乳腺發育 3 二、類胰島素生長因子 7 (一)IGFs生成與重要性 7 (二)IGFs之功能 8 (三)生長激素及IGF-1於乳腺發育之調控 9 三、大腸桿菌表現重組蛋白質系統(pET system) 14 (一)目標基因表現 14 (二)目標蛋白質純化 15 研究目的 16 pcDNA4B/myc-His 質體DNA構築 16 pLKO AS3w.puro及pLKO AS3w.tGFP質體DNA構築 16 pET15b及pET28a質體DNA構築 17 材料與方法 18 一、山羊IGF-1 cDNA之選殖 18 (一)組織total RNA萃取 18 (二)反轉錄酶聚合鏈反應(reverse transcription polymerase chain reaction, RT-PCR) 18 (三)去氧核糖核酸聚合酶鏈反應(polymerase chain reaction, PCR) 19 二、質體pGEM-T easy-gIGF-1與pcDNA4-gIGF-1 myc/His 架接 20 (一)質體pGEM-T easy-gIGF-1架接 20 (二)細菌電穿孔轉型(electroporation transformation)及X-gal藍白菌落篩選 21 (三)質體DNA小量萃取(DNA mini-extraction) 22 (四)質體DNA中量萃取(DNA midi-extraction) 23 (五)質體pcDNA4-gIGF-1 myc/His 架接 24 三、山羊乳腺上皮細胞株(caprine mammary gland epithelial cell line, CMC)培養 26 (一)CMC培養條件 26 (二)CMC解凍活化 26 (三)CMC繼代培養 27 四、於CMC細胞內表現山羊IGF-1之偵測 27 (一)CMC細胞轉染(transfection) 27 (二)細胞蛋白質萃取及其定量分析 28 (三)細胞免疫螢光染色(immunostaining) 31 結果 33 一、山羊IGF-1 cDNA選殖 33 二、外源性山羊IGF-1於CMC細胞株中之表現 34 討論 36 一、山羊IGF-1 cDNA序列分析及載體之構築 36 二、山羊IGF-1於乳腺細胞中之表現位置 36 三、構築慢病毒表現載體系統 36 四、構築大腸桿菌表現重組蛋白質載體系統 37 結論 38 參考文獻 51 附錄 63 附錄 1. 第一型類胰島素生長因子於各物種間之比較。 63 附錄 2. 選殖之山羊IGF-1與其結合蛋白及受器之結合區域預測結果。 64 附錄 3. 乳腺發育過程之上皮與基質細胞交互作用示意圖。 65 附錄 4. 乳腺雛型圖。 66 附錄 5. 小鼠胚乳腺組織生成流程圖。 66 附錄 6. 末端芽形態。 67 附錄 7. 全身內分泌與乳腺組織旁泌之GH對IGF-1產製於乳腺發育之影響概述。 67 附錄 8. 山羊IGF-1 cDNA架構至慢病毒表現系統質體pLKO AS3w.puro流程圖。 68 附錄 9. 山羊IGF-1 cDNA架構至慢病毒表現系統質體pLKO AS3w.tGFP流程圖。 70 附錄 10. 密碼子選擇後成熟態山羊IGF-1 cDNA架構至大腸桿菌表現重組蛋白質系統質體pET15b流程圖。 72 附錄 11. 山羊IGF-1 cDNA架構至大腸桿菌表現重組蛋白質系統質體pET28a流程圖。 74 附錄 12. 結合Gas7基因且經密碼子選擇後之成熟態山羊IGF-1 cDNA架構至大腸桿菌表現重組蛋白質系統質體pET15b流程圖。 76 | |
dc.language.iso | zh-TW | |
dc.title | 選殖並表現山羊第一型類胰島素生長因子於乳腺上皮細胞 | zh_TW |
dc.title | Cloning and expression of type 1 insulin-like growth factor in caprine mammary gland epithelial cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.advisor-orcid | ,姜延年(ynjiang@ntu.edu.tw) | |
dc.contributor.oralexamcommittee | 楊瀅臻(Ying-Chen Yang) | |
dc.subject.keyword | 乳腺上皮細胞, | zh_TW |
dc.subject.keyword | IGF-1,mammary epithelial cell,protein expression,mammary gland development, | en |
dc.relation.page | 77 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-19 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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