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研究生:廖郁文
研究生(外文):Yu-Wen Liao
論文名稱:非酯化脂肪酸與 β-羥基丁酸對脂多醣刺激之牛多形核嗜中性球免疫調節影響
論文名稱(外文):Effects of non-esterified fatty acids and β-hydroxybutyric acid on lipopolysaccharides induced bovine polymorphonuclear neutrophils immunomediation
指導教授:王建鎧
指導教授(外文):Chien-Kai Wang
口試委員:陳淵國陳洵一
口試日期:2020-01-15
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:2020
畢業學年度:108
語文別:中文
論文頁數:76
中文關鍵詞:乳牛轉換期負能量平衡非酯化脂肪酸β-羥基丁酸多形核嗜中性球細胞激素
外文關鍵詞:Dairy cattletransition periodnegative energy balancenon-esterified fatty acidβ- hydroxybutyric acidpolymorphonuclear neutrophilcytokines
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乳牛於分娩前後三週之轉換期 (Transition period) 階段,經歷懷孕末期胎兒快速發育階段、分娩及開始進入泌乳初期的大量耗能,加以乾物質採食量下降,能量攝取不足,而產生負能量平衡 (Negative energy balance, NEB) 現象。為彌補此時的過度消耗將動員體脂,釋出大量可供利用的非酯化脂肪酸 (Non-esterified fatty acid, NEFA),在快速能量需求下, NEFA 不完全氧化、走向酮體生成,產生 β-羥基丁酸 (β- hydroxybutyric acid, BHBA),在分娩後明顯上升,持續過長時間或過於劇烈時將誘發代謝性疾病,並可能進一步影響免疫功能,提高發炎相關疾病的發生率。高濃度 NEFA 可能透過活化牛多形核嗜中性球 (Polymorphonuclear neutrophil, PMN) 表面受體,進而提升促發炎細胞激素表現與氧化壓力,然而 BHBA 部分僅有對 PMN 吞噬能力、噬菌能力影響之研究,其對免疫調節相關細胞激素表現之影響仍未有探討。本研究旨在透過體外試驗,探討乳牛於轉換期間,血漿中高濃度之 NEFA 及 BHBA 對荷蘭牛 PMN 免疫活性之影響,並以脂多醣 (Lipopolysaccharides, LPS) 作為感染刺激因子。經頸靜脈採集健康懷孕乾乳牛周邊血,分離出 PMN,以 0.1、0.6、1.5 mM 之 NEFA,2、5 mM 之 BHBA 分別培養 2 小時,並分為控制組 (Control) 與 LPS 刺激組 (1μg/mL),再利用即時聚合酶連鎖反應分析其細胞激素表現量,並針對經培養之 PMN 存活率、細胞粒徑變化、細胞核形態變化進行觀察。試驗採樣牛隻 (n=7) 血清中 NEFA 平均濃度為 0.09 ± 0.01 mM,BHBA 濃度則為 0.39 ± 0.02 mM,顯示牛隻於採樣前均處於非 NEB 狀態。細胞體外試驗部分,在 LPS 刺激下,1.5 mM 之 NEFA 處理相較於無添加組提升介白素 6 之表現 (2.63倍),介白素 1β、介白素 8 有上升趨勢,腫瘤壞死因子 α 則在高濃度 NEFA 處理下受到抑制。在 BHBA 處理下則無明顯變化。PMN 之細胞存活率、細胞粒徑、細胞核形態在 NEFA 與 BHBA 處理下皆無明顯差異。本研究顯示,相較於 BHBA,NEFA 可能為乳牛轉換期間對 PMN 的主要影響因子,並可能在感染刺激下對 PMN 的免疫活性造成進一步影響。
During periparturient transition period of dairy cows, insufficient dry matter intake, rapid growth of fetus at final stage, and preparation of milk production tend to a negative energy balance (NEB) status. Non-esterified fatty acids (NEFA) are released due to lipid mobilization in the adipose tissue due to a metabolic shift to ketoneogenesis for a rapid ATP supply, and β-hydroxybutyric acid (BHBA), the incomplete oxidation product of NEFA, also increases drastically after parturition. High levels of NEFA increase the production of inflammatory-related cytokines and oxidative stress of polymorphonuclear neutrophil (PMN) by activating receptors. However, the immunomodulatory effects of cytokines by BHBA and NEFA are still not clear. This study utilized PMN under lipopolysaccharides (LPS) stimulation to mimic infectious conditions to investigate the effects by NEFA and BHBA. PMNs were isolated from jugular vein blood of dry-period pregnant cows then treated with NEFA (0.1, 0.6, and 1.5 mM) or BHBA (2 and 5 mM) for 2 hours in combination with LPS (1μg/mL). The transcription levels of IL1β, IL6, IL8, IL10 and TNFα were analyzed by qPCR. Viability, diameter and morphology of PMN were also determined. The concentration of serum NEFA (0.09 ± 0.01 mM) and BHBA (0.39 ± 0.02 mM) of the sampling cows (n=7) suggested a normal condition, not under NEB. The expression of IL6 elevated (2.63 folds) when treated with 1.5 mM NEFA under LPS stimulation. In contrast to the LPS alone group, the expression of IL1β and IL8 were increased but TNFα was suppressed at higher NEFA concentrations. BHBA had no effects on cytokine expressions. Viability, diameter and morphology of PMN showed no changes by NEFA and BHBA treatment. In conclusion, high levels of blood NEFA in transient cows alter immune functions of PMNs rather than BHBA.
目次
中文摘要 i
英文摘要 ii
目次 iii
圖次 vi
表次 viii
壹、前言 1
貳、文獻探討 2
一、乳牛轉換期 (Transition period) 之生理變化 2
(一) 乳牛於轉換期之負能量平衡狀態 (Negative energy balance, NEB) 2
(二) 乳牛於轉換期之能量代謝變化 2
(三) 乳牛於轉換期之內泌素變化 3
二、脂質動員與其代謝物對轉換期乳牛之影響 6
(一) 非酯化脂肪酸 (Non-esterified fatty acid, NEFA) 6
(二) β-羥基丁酸 (β-hydroxybutyric acid, BHBA) 7
(三) NEFA、BHBA 與免疫相關疾病之關聯 8
三、乳牛周邊血白血球種類與功能 8
(一) 免疫系統簡介 8
(二) 牛周邊血中之白血球種類 9
(三) 多形核嗜中性球 (Polymorphonuclear neutrophil, PMN) 之功能 11
(四) 發炎相關細胞激素 12
1. 介白素 1β (Interleukin 1 beta, IL1β) 13
2. 介白素 6 (Interleukin 6, IL6) 13
3. 介白素 8 (Interleukin 8, IL8) 14
4. 介白素 10 (Interleukin 10, IL10) 14
5. 腫瘤壞死因子α (Tumor necrosis factor alpha, TNFα) 14
四、乳牛轉換期之脂質動員及代謝產物變化對其體內細胞激素調控 15
參、材料與方法 17
一、試驗動物之選用及樣品採集 17
二、血清之分離、保存、測定 17
三、In vitro 之 PMN 細胞模型製備 18
四、細胞粒徑、存活率測定 20
五、細胞固定與劉氏染色抹片製作 20
六、PMN之培養與處理 20
(一) LPS 配置、分裝、保存 21
(二) BHBA與LPS刺激 21
(三) NEFA與LPS刺激 22
1. NEFA stock配置 22
2. NEFA處理培養基配置 24
(四) 細胞與培養基回收 25
七、PMN之細胞 cytokine mRNA 表現量測定 26
(一) Total RNA之萃取、品質、完整度測試 26
1. Total RNA 之萃取 26
2. Total RNA 完整度測試 27
(二) 反轉錄反應 (Reverse Transcription, RT) 28
(三) 引子設計 29
(四) 即時聚合酶連鎖反應 (Real-time polymerase chain reaction, qPCR) 31
八、統計方法 32
肆、結果 33
一、試驗牛隻血清中 NEFA、BHBA 濃度 33
二、PMN 細胞培養後之存活率與細胞粒徑變化 34
(一) 細胞經 BHBA 處理培養 2 小時後之存活率與細胞粒徑變化 34
(二) 細胞經 NEFA 處理培養 2 小時後之存活率與細胞粒徑變化 37
三、PMN 細胞培養後之細胞核形態變化 40
四、BHBA 處理對 PMN cytokines mRNA 表現量之影響與表現倍率 43
五、NEFA 處理對 PMN cytokine mRNA 表現量之影響與表現倍率 47
伍、討論 51
一、PMN 採樣策略與培養模式之建立 51
(一) 試驗牛隻血清中 NEFA、BHBA 濃度 51
(二) PMN 細胞培養後之存活率與細胞粒徑變化 51
(三) PMN 細胞培養後之細胞形態變化 52
二、BHBA 處理對 PMN cytokine mRNA 表現量之影響 53
三、NEFA 處理對 PMN cytokine mRNA 表現量之影響 55
四、轉換期發炎相關細胞激素調節與乳牛免疫功能轉變之可能關聯 62
陸、結論 66
柒、參考文獻 68
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