中文摘要

在動情周期中，動情素的上升使得子宮內膜增生，黃體素則刺激子宮內膜腺體增生，讓內膜變的肥厚且富含水份，形成良好的環境以利胚胎的著床。然而，在胚胎著床到生產的114天中，卻有40% 的胚胎死亡，而大部分發生於懷孕的10-30天，因此推測母體在懷孕前的狀況，會影響到母豬的生產率。因此本研究想找出是否有較具指標性的生物標記 (biomarker)，能用來判斷母體的身體狀況是否適合進行受孕，進而提高母豬的生產率。IGFs (insulin-like growth factors) 為一種和胰島素具有高度序列相似度的蛋白質，它能夠促進細胞的增生及減少細胞凋亡，使個體成長。而IGFs在血液中需要IGFBP3 (insulin-like growth factor binding protein 3) 的輔助，來延長IGFs在血液中的半生期，並且透過改變IGFBP3的活性，能調節IGFs在血液中的濃度。過去的研究顯示IGFBP3可能不利於濾泡的成長，進而影響母豬的產率，因此了解血液中IGFBP3的表現，也許對了解母豬的生產情況有相關性。本實驗利用biotin-streptavidin的呈色系統，偵測IGFBP3，在43 kDa偵測到IGFBP3的色帶。而在分析IGFBP3含量與產率的實驗中發現，在低產豬的血液中，IGFBP3的濃度較高，由此結果推測，IGFBP3可能透過與IGF受器競爭IGF，使得IGF與受器的結合下降，進而降低細胞增生。在低產豬中，因血液中IGFBP3的量較高，降低了IGF與受體的結合，無法促使細胞的增生，而使產率下降。此結果顯示IGFBP3可能是具有指標性的生物標記 (biomarker)，能用來判斷母體的身體狀況是否適合進行受孕。

Abstract

In female, estrogen and progesterone stimulated endothelium growth and endometrial glands secretion respectively. Estrogen and progesterone let placenta become moist and establish an optimal environment for the implantation. However, up to 40% piglets were lost between pregnant period, and most of fetal losses clustered from gestation day 10-30. Therefore, physical condition of pre-pregnancy might play a part in reproductive rate. This study wanted to find out biomarkers to estimate whether sow physical condition is suitable for mating. IGFs (Insulin-like growth factors) can stimulate cell proliferation and decline apoptosis, and the protein sequences of IGFs are very similar to that of insulin. Unlike insulin, IGFs forme a complex with IGFBPs (insulin-like growth factor binding proteins) in blood circulation, this interaction could prolong the half-life of IGFs. Altering IGFBP3 activity also can regulate the concentration of IGFs in blood. Previous studies indicated that IGFBP3 might inhibit follicles growth and then affected sow reproductive rate. Thus IGFBP3 might be a potential biomarker to realize whether sow physical condition is suitable for mating. We used biotin-streptavidin system to detect the IGFBP3 band at 43 kDa. Analysis of IGFBP3 expression showed that IGFBP3 concentration in low reproductive sow was higher than that in high reproductive sow. The result suggests that the competition between IGFBP3 and IGFs receptor for IGF is a critical regulation mechanism. When IGFBP3 concentration is high in blood, it will interact with IGF, which reduces the interaction between IGFs and IGFs receptor. As a result, it will decline cell proliferation and reproductive rate in sow. In conclusion, IGFBP3 could be a potential biomarker for reproductive rate prediction.

目次

中文摘要 ….. i
Abstract ..... ii
縮寫表 ..... iii
第一章 緒論 ……. 1
1.1 動情週期 (estrous cycle) ……. 1
1.2 胚胎發育與著床 (embryo development and implantation) ……. 2
1.3 類胰島素生長因子系統 (IGF system or Insulin-like growth factor system) ……. 4
1.3.1 The GF-IGF axis ……. 4
1.3.2 類胰島素生長因子 (IGF or Insulin-like growth factor) ……. 5
1.3.3 類胰島素生長因子受器 (IGF-R or Insulin-like growth factor receptor) ……. 6
1.3.3.1 IGF-1R (insulin-like growth factor-1 receptor) ……. 6
1.3.3.2 IGF-2R (insulin-like growth factor-2 receptor) ……. 7
1.3.4 類胰島素生長因子結合蛋白 (IGFBPs or Insulin-like growth factor binding proteins) ……. 7
1.4 IGFBP3與懷孕的相關性 ……. 9
1.5 研究動機 9
第二章 材料與方法 ……. 12
2.1 血液樣本 ……. 12
2.2 樣本製備 ……. 12
2.2.1 去除免疫球蛋白G (IgG) ……. 12
2.2.2 去除白蛋白 (albumin) ……. 12
2.2.3 膠體過濾法 ……. 12
2.2.4 酒精沉澱 ……. 12
2.2.5 甲醇-氯仿沉澱 ……. 13
2.2.6 蛋白質定量 ……. 13
2.3 一般電泳檢驗法 ……. 13
2.3.1 SDS 膠體電泳 ……. 13
2.3.2 二維電泳 (two-dimensional electrophoresis) ……. 13
2.4 蛋白質呈色法 ……. 14
2.4.1 CBR (Coomassie Brilliant Blue R-250) 染色法 ……. 14
2.4.2 免疫呈色 ……. 14
2.4.2.1 Biotinylated IGF-II與 biotinylated anti-IGFBP3 Ab的製備 ……. 14
2.4.2.2 蛋白質轉印 ……. 14
2.4.2.3 免疫呈色 ……. 15
2.5 膠體內蛋白酶水解 ……. 15
第三章 結果與討論 ……. 16
3.1 利用2-DE進行高低產分析 ……. 16
3.2 樣本的純化 ……. 16
3.2.1 親和層析法 ……. 16
3.2.2 膠體層析法 ……. 17
3.3 酒精沉澱與biotin-streptavidin呈色 ……. 17
3.4 找尋血液中IGFBP3含量與高低產間的相關性 ……. 18
3.5 高低產預測 ……. 19
圖與表 ……. 20
第四章 結論 ……. 28
參考文獻 ……. 29
問與答 ……. 32

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