Studies on immunomodulatory functions of lactoferrin under inflammatory stress in vitro and in vivo

乳鐵蛋白是一種吸附鐵之醣蛋白，存在於哺乳動物的乳汁和外分泌腺中，研究指出乳鐵蛋白具有抗菌、抗癌、調節免疫…等多種生理活性。因此本篇研究以體外不同實驗模式下，探討重組人類乳鐵蛋白和牛乳鐵蛋白之免疫調節功效，並進一步以脂多醣 (lipopolysaccharide, LPS) 經腹腔注射誘發發炎之動物模式，探討牛乳鐵蛋白免疫調節功能。 在體外細胞培養實驗結果顯示，牛乳鐵蛋白在濃度31.3和125 μg/ml有顯著抑制脾臟細胞增生現象，但與乳鐵蛋白吸附鐵離子的能力無關。重組人類乳鐵蛋白在高濃度(1000及1250μg/ml)下，有增加腹腔細胞存活之趨勢，推測重組人類乳鐵蛋白能活化腹腔細胞。牛和重組人類乳鐵蛋白對發炎脾臟及腹腔細胞之增生與存活皆無顯著影響。在不同實驗模式下，重組人類乳鐵蛋白濃度在250 μg/ml下對發炎媒介物(IL-1β、IL-6、IL-10、TNF-α)有顯著增加，而牛乳鐵蛋白則無。 在體內實驗中，小鼠經餵食牛乳鐵蛋白4週後，結果顯示餵食高濃度(2.55 mg/mouse)乳鐵蛋白會降低小鼠血液中Ig A、Ig M和Ig G濃度，但增加Ig E濃度，推測餵食高濃度(2.55 mg/mouse)牛乳鐵蛋白4週可能使個體產生過敏傾向；但對腹腔注射LPS誘發發炎小鼠之腹腔及脾臟細胞所分泌的發炎媒介物則無顯著影響，由本實驗結果顯示在餵食牛乳鐵蛋白4週後，在腹腔注射LPS誘發發炎的動物實驗模式下，對小鼠的發炎情況無顯著改善作用。Lactoferrin is an iron-binding glycoprotein which exists in milk as well as other exocrine secretion in mammals. In recent years the study has indicated that lactoferrin might have multiple physiological functions such as antimicrobial, anti-tumor and immunomodulatory activities.To evaluate the effects of immounmodulatory, bovine and recombinant lactoferrin were first chosen to subject to different in vitro experimental models in this study. In an in vivo test, bovine lactoferrin was further investigated its immunomodulatory effects on lipopolysaccharide (LPS)-induced (i.p.) inflammation of mouse model. The results showed that bovine lactoferrin significantly inhibited splenocyte proliferation at concentrations of 31.3 and 125 μg/mL. However, the inhibitory function of lactoferrin was irrelevant to its iron absorption capacity. Recombinant human lactoferrin slightly increased cell viability of peritoneal cells at higher concentrations (1000 and 1250 μg/ml), suggesting that recombinant human lactoferrin might activate peritoneal cells. However, both bovine and recombinant human lactoferrin did not significantly affect the cell viability of inflamed splenocytes and peritoneal cells. Recombinant human lactoferrin (250 μg/ml) demonstrated a significant increased effect on inflammatory mediators (IL-1β、IL-6、IL-10、TNF-α) under different experimental models, but bovine lactoferrin did not. The results from in vivo study showed that bovine lactoferrin administration for 4 weeks decreased serum Ig A, Ig M and Ig G levels, but increased Ig E level at high dose feeding group. The results suggest that feeding with high concentrations (2.55 mg/ mouse) of lactoferrin for long term may induce allergic tendencies in vivo. Bovine lactoferrin administration did not significantly affect peritoneal cells and splenocytes from LPS-induced inflammation mice. In conclusion, bovine lactoferrin administration showed no significant improvement effects on LPS-induced systemic inflammation.表目錄…….........................................................................................................V 圖目錄………………………………………………………………………VII 縮寫對照表........................................................................................................IX 中文摘要.............................................................................................................X Abstract ............................................................................................................ XI 第一章 緒言........................................................................................................1 第二章 文獻回顧...............................................................................................2 第一節 免疫系統介紹................................................................................2 壹、免疫系統(immune system) ...........................................................2 一、先天性免疫反應(innate immune response)........................................2 二、適應性免疫反應(adaptive immune response)....................................3 貳、細胞激素(cytokines) .....................................................................6 一、一般性質.............................................................................................6 二、各種細胞激素介紹.............................................................................6 參、發炎與C反應蛋白(C-reactive protein).........................................8 一、發炎.....................................................................................................8 二、C反應蛋白(C-reactive protein) .........................................................11 肆、抗體.............................................................................................11 第二節 內毒素..........................................................................................13 第三節 乳鐵蛋白......................................................................................14 壹、簡介.............................................................................................14 貳、結構.............................................................................................15 參、安全性.........................................................................................17 肆、重組蛋白.....................................................................................17 伍、乳鐵蛋白在免疫上角色.............................................................17 第四節 乳鐵蛋白生理活性......................................................................19 壹、抗病毒.........................................................................................19 貳、抗腫瘤.........................................................................................19 參、抗菌.............................................................................................20 肆、抗發炎.........................................................................................23 伍、免疫調節.....................................................................................23 第五節 研究動機及目的..........................................................................25 第三章 牛及重組人類乳鐵蛋白體外調節免疫之功能影響.........................27 第一節 前言..............................................................................................27 第二節 材料與方法..................................................................................28 壹 初代免疫細胞之取得及培養.....................................................28 貳、脾臟細胞增生與腹腔細胞存活活性分析.................................31 參、細胞激素測定.............................................................................32 肆、一氧化氮(NO)之測定.................................................................34 伍、統計分析.....................................................................................35 第三節 結果..............................................................................................36 壹、牛和重組人類乳鐵蛋白對BALB/c雌鼠脾臟細胞增生的影響 .............................................................................................................36 貳、牛和重組人類乳鐵蛋白對BALB/c雌鼠腹腔細胞存活的影響 .............................................................................................................36 参、牛和重組人類乳鐵蛋白對BALB/c雌鼠經LPS誘發的發炎脾臟 細胞增生的影響.................................................................................41 肆、牛和重組人類乳鐵蛋白對BALB/c雌鼠經LPS誘發的發炎腹腔 細胞存活的影響.................................................................................41 伍、牛和重組人類乳鐵蛋白在以LPS刺激的實驗模式下對BALB/c 雌鼠脾臟細胞TH1 和TH2 細胞激素分泌的影響..............................46 陸、牛和重組人類乳鐵蛋白在以LPS刺激的實驗模式下對BALB/c 雌鼠腹腔細胞發炎媒介物分泌的影響.............................................46 柒、不同濃度乳鐵蛋白下添加不同濃度鐵離子對脾臟細胞增生影 響.........................................................................................................56 捌、牛和重組人類乳鐵蛋白在不同濃度下對BALB/c 雌鼠脾臟細 胞對IL-2 分泌的影響.........................................................................56 第四節 討論..............................................................................................62 第五節 結論..............................................................................................63 第四章 小鼠經腹腔注射LPS誘發發炎狀態不同時間點之變化..................64 第一節 前言..............................................................................................64 第二節 材料與方法..................................................................................64 壹、實驗動物飼養.............................................................................64 貳、血清收集及分析.........................................................................64 參、細胞之取得及培養.....................................................................64 肆、細胞激素測定.............................................................................65 伍、一氧化氮(NO)之測定.................................................................65 陸、血清中急性發炎蛋白C反應蛋白測定(C-reactive protein) ......65 柒、統計分析.....................................................................................66 第三節 結果..............................................................................................67 壹、腹腔注射LPS對BALB/c雌鼠體重的影響................................67 貳、BALB/c小鼠經腹腔注射LPS後時間點之組織與相對組織重的 影響.....................................................................................................67 参、腹腔注射LPS對BALB/c雌鼠脾臟和腹腔細胞數的影響........67 肆、BALB/c小鼠經腹腔注射LPS後不同時間點對脾臟細胞分泌發 炎媒介物之影響.................................................................................72 伍、BALB/c小鼠經腹腔注射LPS後不同時間點對腹腔細胞分泌發 炎媒介物之影響.................................................................................72 陸、BALB/c小鼠經腹腔注射LPS後不同時間點對血清中急性發炎 蛋白與IL-6 之影響.............................................................................72 第四節 討論..............................................................................................83 第五節 結論..............................................................................................84 第五章 牛乳鐵蛋白對腹腔注射LPS誘發發炎BALB/c小鼠之免疫反應影響 ...........................................................................................................................85 第一節前言................................................................................................85 第二節 材料與方法..................................................................................85 壹、實驗動物飼養.............................................................................85 貳、材料.............................................................................................85 參、實驗設計操作模式.....................................................................86 肆、血清收集及分析.........................................................................88 伍、細胞之取得及培養.....................................................................89 肆、細胞激素測定.............................................................................89 陸、一氧化氮(NO)之測定.................................................................89 柒、血清中急性發炎蛋白C反應蛋白測定(C-reactive protein,CRP) .............................................................................................................89 捌、統計分析.....................................................................................90 第三節 結果..............................................................................................91 壹、餵食不同濃度牛乳鐵蛋白對BALB/c雌鼠體重變化...............91 貳、餵食不同濃度牛乳鐵蛋白對脂多醣腹腔注射BALB/c雌鼠腹腔 細胞之組織重及其相對重量的影響.................................................91 參、餵食不同濃度牛乳鐵蛋白對以脂多醣腹腔注射之BALB/c雌鼠 脾臟與腹腔細胞分泌發炎媒介物之影響.........................................91 肆、餵食不同濃度牛乳鐵蛋白以脂多醣腹腔注射之BALB/c雌鼠血 清中發炎媒介物之影響.....................................................................91 伍、餵食牛乳鐵蛋白對BALB/c雌鼠血清非特異性抗體濃度變化 .............................................................................................................98 陸、餵食不同濃度牛乳鐵蛋白對以脂多醣腹腔注射之BALB/c雌鼠 血清抗體之影響.................................................................................98 第四節 討論............................................................................................104 第五節 結論............................................................................................104 第六章 總結論................................................................................................106 第七章 參考文獻...........................................................................................10