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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 姜延年,朱有田 | |
dc.contributor.author | Chien-Yi Hung | en |
dc.contributor.author | 洪千翊 | zh_TW |
dc.date.accessioned | 2021-06-07T23:48:52Z | - |
dc.date.copyright | 2014-03-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-02-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16885 | - |
dc.description.abstract | 對哺乳動物而言,乳腺是個特殊且重要的器官,乳腺發育也與乳用動物的經濟效益息息相關。出生後哺乳動物的乳腺發育可分為幼年期、發身期、懷孕期、泌乳期、退乳期,每時期皆受許多不同的激素與生長因子調控,其中第一型類胰島素生長因子 (insulin-like growth factor 1, IGF-1) 對發身期乳管發育、懷孕後期及泌乳期的乳腺泡增生皆有重大影響。一般而言,IGF-1可透過PI3K/Akt或MAPK訊息傳遞路徑,調控細胞增生、存活、分化。過去研究發現IGF-1在小鼠乳腺組織的mRNA表現量於泌乳時期急遽上升,顯示IGF-1對乳腺分化可能有一定的重要性。乳蛋白如beta-酪蛋白表現為乳腺分化的指標之一,而研究發現IGF-1與生乳激素同時處理小鼠乳腺上皮細胞時,可誘導beta-酪蛋白之表現。然而過去在IGF-1對乳腺作用之研究皆著重以囓齒類動物為模式,對於IGF-1在乳用動物之乳腺之作用較無深入探討。因此本研究利用不朽化山羊乳腺上皮細胞 (caprine mammary epithelial cells, CMECs),探究IGF-1對發身時期乳腺上皮細胞發展之影響,以及探討IGF-1對泌乳時期乳腺上皮細胞分化之影響。
首先以Matrigel體外培養CMECs三維立體乳腺泡,使用含有皮質酮 (hydrocortisone)、表皮生長因子 (epidermal growth factor)、霍亂毒素 (cholera toxin)、馬血清 (horse serum)、胎牛血清 (fetal bovine serum, FBS),及不同濃度重組IGF-1之171培養液。培養12天後發現添加50、100、200 ng/mL IGF-1的組別,比未添加IGF-1的組別,乳腺泡生成數量顯著較多且半徑長度顯著較大 (P < 0.05);然而在50、100、200 ng/mL IGF-1的組別之間,乳腺泡生成數量及半徑長度則無顯著差異。接著將CMECs培養於含有2% FBS及不同濃度IGF-1之171培養液內,48小時後以MTT法偵測細胞增生,發現添加50 ng/mL及200 ng/mL IGF-1之組別,細胞增生程度顯著較未添加IGF-1之組別高 (P < 0.05)。為瞭解IGF-1是否促進細胞存活,將CMECs血清飢餓12小時後,加入IGF-1使最終濃度為50 ng/mL,30分鐘後收取蛋白質並進行西方墨點法分析,發現蛋白質激酶B (protein kinase B, Akt) 磷酸化現象較未添加IGF-1高。而為瞭解IGF-1對乳蛋白表現的影響,將穩定表現beta-酪蛋白啟動子驅動螢火蟲冷光素酶報導基因的山羊乳腺上皮細胞培養三天後,以含有泌乳素、皮質酮及不同濃度IGF-1之分化培養液誘導報導基因表現,結果在添加50 ng/mL IGF-1組別測得beta-酪蛋白啟動子活性約為未添加IGF-1組別的四倍 (P < 0.05),但50 ng/mL與400 ng/mL兩組之間則沒有顯著差異;而使用西方墨點法分析,發現添加IGF-1的組別,其訊息傳導及轉錄活化因子5a (signal transducer and transcription activator 5a, Stat5a) 不受影響,而Erk及Akt的磷酸化程度皆比未添加IGF-1組別高。進一步在分化培養液添加PI3K之抑制劑 (LY294002),結果發現降低Akt磷酸化並不顯著影響IGF-1促進之beta-酪蛋白表現。 綜上所述,IGF-1可促進發身時期CMECs之增生及存活,使乳腺泡數量及大小增加。當生乳激素存在時,IGF-1可促進CMECs之beta-酪蛋白表現,且此現象可能不是透過PI3K/Akt訊息傳遞路徑所誘導。然而IGF-1對促進beta-酪蛋白之表現是透過何種訊息傳遞路徑,未來仍需進一步試驗證實。 | zh_TW |
dc.description.abstract | The development of mammary gland is regulated by many different hormones and growth factors, and insulin-like growth factor 1 (IGF-1) is one of which an important factor to duct development during puberty. Generally, IGF-1 mediates cell proliferation, survival, and differentiation through PI3K/Akt and MAPK signaling pathways. Previous studies showed that the mRNA level of IGF-1 in mouse mammary glands increases significantly during lactation, indicating IGF-1 might play a role in mammary gland differentiation, which results in milk protein expression. It was found that IGF-1, along with lactogenic hormones, significantly enhances beta-casein expression in mouse mammary epithelial cells. However, previous studies were mostly based on rodents. The role of IGF-1 in mammary glands of dairy animals is still not comprehensively studied. In this study, caprine mammary epithelial cells (CMECs) were used to investigate (1) the function of IGF-1 in the development of mammary epithelial cells during puberty, and (2) the function of IGF-1 in differentiation of mammary epithelial cells during lactation.
The three-dimensional culture of CMECs was grown in 171 medium containing Matrigel and IGF-1 to observe the effect of IGF-1 on acini formation. After 12-day incubation, the amount and size of acini in groups with IGF-1 were found to be significantly larger and bigger than the group without IGF-1 (P < 0.05). MTT assay was then used to assess whether cell proliferation is increased in the presence of IGF-1. CMECs were incubated with 2% FBS and different concentration of IGF-1 for 48 h, and MTT assay was performed. Results showed that cells in groups with IGF-1 were more viable than the group without IGF-1 (P < 0.05). To understand whether IGF-1 promotes cell survival, CMECs were incubated in serum-free medium for 12 h, and IGF-1 was then added. Lysates were collected after 30 min and western blotting was performed to detect the survival signal, phosphorylation of Akt. It was found that phosphorylation of Akt was enhanced when IGF-1 was added. To understand the effect of IGF-1 on differentiation, CMECs stably expressing the beta-casein promoter-driven luciferase reporter gene were grown to confluent, and then induced with differentiation medium containing lactogenic hormones and IGF-1. Results showed that the activity of beta-casein promoter in the group with IGF-1 was about 4-fold higher than the group without IGF-1 (P < 0.05). Western blotting was further performed to investigate which signaling pathway might be involved in the IGF-1-enhanced beta-casein expression. It was found that Akt phosphorylation was dramatically accumulated when beta-casein expression is enhanced by IGF-1. CMECs were then induced with additional PI3K inhibitor, LY294002, and it was found that decreased phosphorylation of Akt does not affect the IGF-1-enhanced beta-casein expression. In conclusion, IGF-1 might increase proliferation and survival of CMECs during puberty, resulting in larger amount and bigger size in acini formation. When lactogenic hormones are present, IGF-1 could enhance beta-casein promoter activity, and this consequence might not be through the PI3K/Akt signaling pathway. However, the precise signaling cascade is still left to be investigated. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T23:48:52Z (GMT). No. of bitstreams: 1 ntu-103-R00626012-1.pdf: 2188222 bytes, checksum: d57d9c2a7aa2c8dbcab079ddc2f8dfbd (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員審定書.....i
謝誌.............ii 中文摘要..........iii 英文摘要..........v 目錄.............vii 圖目錄............viii 表目錄............ix 壹、前言..........1 貳、文獻檢討.......2 參、材料與方法.....12 肆、結果..........20 伍、討論..........24 陸、結論..........29 參考文獻..........40 附錄.............51 | |
dc.language.iso | zh-TW | |
dc.title | 第一型類胰島素生長因子於山羊乳腺上皮細胞之功能研究 | zh_TW |
dc.title | The functional study of IGF-1 in caprine mammary epithelial cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃木秋,徐濟泰 | |
dc.subject.keyword | 乳腺泡,beta-酪蛋白,山羊,第一型類胰島素生長因子,乳腺上皮細胞, | zh_TW |
dc.subject.keyword | acini,beta-casein,caprine,IGF-1,mammary epithelial cells, | en |
dc.relation.page | 56 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-02-14 | |
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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