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研究生:荷琇媛
研究生(外文):Xiu-Yuan He
論文名稱:性固醇類內泌素於牛子宮內膜上皮細胞對生長因子表現之調控
論文名稱(外文):Expression of growth factors regulated by sex steroids in bovine endometrial epithelial cells
指導教授:王建鎧
口試委員:唐品琦陳珠亮
口試日期:2021-07-26
學位類別:碩士
校院名稱:國立中興大學
系所名稱:動物科學系所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:75
中文關鍵詞:雌二醇孕酮生長因子子宮內膜上皮細胞
外文關鍵詞:EstradiolProgesteroneGrowth factorsEndometrial epithelial cell
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胚著床失敗是乳牛懷孕失敗的主要原因之一。在黃體期牛隻子宮內膜細胞主要 受到雌二醇 (Estradiol,E2) 和孕酮 (Progesterone,P4) 所調控並進行一系列的增生 與分化,在這些內泌素之調控下子宮內膜功能的建立將對後續的著床造成影響。E2在黃體前期會出現一次濃度較低的潮湧並維持著一定濃度和P4共同影響子宮中的 環境,但其生理機制目前尚未明朗。牛子宮內膜上皮細胞 (Bovine endometrial epithelial cells, bEECs) 之分泌作用對懷孕前期子宮內膜之建立很重要 ,而E2會刺 激子宮內膜上皮之增生,其作用會受到P4抑制並且促進腺體上皮細胞之分化。除此之外,這些性固醇類內泌素在牛的黃體期當中會透過刺激生長因子 (Growth factors) 分泌來對子宮內膜細胞進行各種調控以利胚著床。因此,本試驗目的為測定E2與P4如何共同調控牛子宮內膜上皮細胞之生長因子表現。本實驗分為兩個部分,試驗一中為測試最理想之基因表現時間點利用體外培養細胞以5、50、1000 pg/ml 之E2刺激16、24、48小時,收樣後測定相關基因表現量。結果顯示Estrogen receptor alpha (ERɑ)、Progesterone receptor (PR) 以及 Fibroblast growth factor 2 (FGF2) 基因之表現在E2的刺激24小時後會有1.4到2倍之提升,反應較16以及48小時顯著 有較佳基因表現。在試驗二中為了釐清性固醇類內泌素對於上皮細胞 中基因表現之調控,利用 5、50 pg/ml E2及5、10 ng/ml P4各別進行刺激以及共 刺激 24 小時,收樣後測定相關基因表現量。研究結果顯示FGF2和Insulin like Growth Factor 1 (IGF1) 在E2 5 pg/ml刺激下分別有1.8及1.9倍的上調,兩者在加入5、10 ng/ml P4共刺激後會出現拮抗作用 ,在蛋白表現的部分也有相似的結果。Vascular endothelial growth factor (VEGFA) 在 E2和P4的刺激之下有1.4- 1.6倍之提升, 並且5 pg/ml E2 + 5 ng/ml P4的共刺激下有趨近於相加 作用之反應。另一方面,PR 在5 pg/ml E2的刺激下有2倍的提升,但共刺激下會受到 10 ng/ml P4的抑制;在加入E2受體抑制劑 (ICI 182,780) 和5 pg/ml E2共同處理後FGF2和Egr 1之基因表現分別受到46及42%的抑制。本研究說明在E2及P4共同處理之下,P4對於子宮內膜上皮細胞中E2上調之FGF2和IGF1表現具有拮抗作用,這顯示在進入黃體期子宮內膜細胞增生、遷移、部分分化以及血管新生會受到限制,而E2及P4上調之VEGFA及EGFR則會促進血管修復以及子宮重塑以促進子宮內膜進入下一個週期。欲釐清生長因子在懷孕前期不同階段中維持正常子宮內膜生理功能之狀態,基質細胞誘導之細胞增生、分化及胚著床對周圍上皮細胞增生、凋亡及遷移之調控需藉由共培養確認,這些研究將協助開發改善早期懷孕成功率之管理策略。
Embryo loss during implantation is one of the main reasons leading to pregnant failure of cattle. During luteal phase, endometrial cells are mainly regulated by Estradiol (E2) and Progesterone (P4) and associated with successful implantation. The second surge of E2 , which appears in the early luteal phase, followed a related low concentration to affect the uterus with P4 , but the physiological mechanism still remained not clear. During luteal phase, E2 and P4 regulated the successful embryo implantation jointly by stimulating the secretion of growth factors in Bovine endometrial epithelial cells (bEECs). The purpose of this study is to investigate how E2 and P4 regulate the growth factor expression in bEECs together. The experiment was divided into two parts. Firstly, bEECs were stimulated with E2 at 5, 50, and 1000 pg/ml for 16, 24, 48 hours. The mRNA expression of growth factors and related genes were measured for determining the proper time of gene expression. The study showed that Estrogen receptor alpha (ERɑ), Progesterone receptor (PR) and Fibroblast growth factor 2 (FGF2) were up regulated 1.4 2 times with E 2 stimulation after 24 hours, which had higher reaction than 16, 48 hours. In second experiment, cells were treated with 5, 50 pg/ml E2 and 5, 10 ng/ml P4 alone or in combination for 24 hours. The aim of the experiment were determined the regulation of hormones on growth factors and related genes. The results showed that FGF2 and Insulin like Growth Factor 1 (IGF1) was up regulated by 5pg/ml E2 about 1.8 1.9 times, which were inhibited after co stimulated with P4. Vascular endothelial growth factor (VEGFA) were induce by sex steroids 1.4 1.6 times, and 5 pg/ml E 2 + 5 ng/ml P 4 had the addition effect . Besides, with the stimulation of 5, 50pg/ml E2, PR was increased 2 times, but the up regulation of E2 was inhibited by 10ng/ml P4. On the other hands, bEECs treated with 5 pg/ml E2 and estradiol receptor inhibitor (ICI 182,780), the gene expression of FGF2 and Egr1 were inhibited about 46 and 42%, respectively. Th e study showed that the antagonism by P 4 which limited the expression of E2 up regulated growth factors : FGF2 and IGF1 , in endometrial epithelial cells , which implied that the proliferation, migration differentiation and angiogenesis in endometrium during the luteal phase was limited. Furthermore, VEGFA and EGFR, which were induced by P4 and E2 might recover blood vessels and remodel the endometrium to prepare continued estrus cycle. Stroma cells regulate proliferation and differentiation of EECs and embryo induces epithelial cell proliferation , apoptosis and migration during implantation . Subsequent experiments on the co cultured test can clarify the expression and function of various growth factors during early pregnancy for successful implantation to improve current fertility in cows.
摘要-i-
Abstract-iii-
目次-v-
表次-viii-
圖次-ix-
壹、前言 - 1 -
貳、文獻探討- 2 -
一、子宮內膜對懷孕維持之影響- 2 -
(一)子宮內膜中的訊號傳遞- 3 -
(二)子宮內膜上皮細胞異常之病理狀態- 4 -
二、子宮內膜之生理變化- 4 -
(一)懷孕前期性固醇類內泌素之調控- 4 -
(二)子宮內膜功能性之影響- 8 -
三、生長因子對子宮內膜之調節- 10 -
(一)纖維母細胞生長因子 (Fibroblast growth factors, FGFs) - 10 -
(二)類胰島素生長因子1 (Insulin-like growth factor 1, IGF1)- 11 -
(三)血管內皮生長因子 (Vascular endothelial growth factor A, VEGFA)- 12 -
(四)轉化生長因子β1 (Transforming growth factor β1, TGFβ1) - 12 -
(五)表皮生長因子受體 (Epidermal growth factor receptor, EGFR)- 13 -
(六)早期生長反應蛋白 (Early growth response protein 1; Egr-1)- 13 -
四、懷孕前期性固醇類內泌素對於細胞內生長因子及相關基因之調控- 14 -
參、材料與方法- 18 -
一、實驗動物以及樣本收集- 18 -
二、細胞增生試驗- 20 -
三、性固醇類內泌素藥物處理試驗- 20 -
(一)理想時間點測試試驗- 21 -
(二)性固醇類內泌素處理試驗- 21 -
四、抑制劑共刺激試驗- 22 -
五、bEECs之細胞mRNA表現量測定- 23 -
(一)Total RNA之萃取、完整度測定- 23 -
(二)反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction, RT-PCR)- 24 -
(三)基因引子序列- 25 -
(四)定量聚合酵素連鎖反應(Quantitative PCR, qPCR)- 27 -
六、西方墨點法 (Western blot)- 28 -
(一)蛋白萃取 (Protein Extraction)- 28 -
(二)蛋白前處理- 29 -
(三)SDS-Page (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)- 30 -
(四)Transfer- 32 -
(五)Blocking- 33 -
(六)一級抗體 (Primary antibody)- 33 -
(七)二級抗體 (Secondary antibody)- 34 -
(八)底片感光(壓片)結果- 35 -
七、酵素免疫分析法 (Enzyme-linked immunosorbent assay;ELISA)- 35 -
八、統計分析- 36 -
肆、結果 - 37 -
一、性固醇類內泌素對牛隻子宮內膜上皮細胞增生之影響 - 37 -
二、不同時間點下雌二醇對ERα、PR以及FGF2之調節- 39 -
三、雌二醇以及孕酮處理24小時後對細胞中相關基因表現之影響- 42 -
四、雌激素受體抑制劑處理24小時後對細胞中生長因子及相關基因表現之影響- 50 -
伍、討論 - 51 -
一、 性固醇類內泌素對子宮內膜上皮細胞增生之影響- 51 -
二、牛隻子宮內膜上皮細胞培養之基因表現最佳時間點- 52 -
三、雌二醇以及孕酮處理24小時後對細胞中相關基因表現之影響- 53 -
(一) 早期懷孕各生長因子之生理意義- 53 -
(二) 性固醇類內泌素透過專一受體對子宮內膜之調節- 56 -
(三) ERα對於生長因子表現之影響- 58 -
四、早期懷孕性固醇類內泌素對上皮細胞分泌作用之調節- 60 -
陸、結論-64-
柒、參考文獻-65-
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