Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98071
標題: 探討乳脂肪球膜對巨噬細胞以及葡聚醣硫酸鈉誘發腸炎之影響
Effect of milk fat globule membrane on macrophages and dextran sodium sulfate-induced colitis
作者: 胡子軒
Tzu-Xuan Hu
關鍵字: 乳脂肪球膜
巨噬細胞
腸道發炎性疾病
milk fat globule membrane
macrophage
intestinal inflammatory disease
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摘要: 乳脂肪球膜 (milk fat globule;MFGM) 為三層磷脂質鑲嵌膜蛋白並包覆三酸甘油酯核心的複雜生物性膜,為維持乳汁中乳脂肪穩定與乳化狀態的重要結構。先前研究已知MFGM鑲嵌之膜蛋白與極性磷脂質成分個別具有許多生物活性之潛力,諸如抑制癌症、降低血膽固醇、促進凋亡細胞吞噬以及促進腸道上皮修復與成熟等功能,並且顯示可能具有免疫調節之功能。因此本研究旨在初步評估整體MFGM分離物對於免疫機能性之影響,以及對於小鼠腸炎之影響。   本研究利用氫氧磷灰石 (hydroxyapatite;HA) 前處理法解離酪乳原料中之酪蛋白微粒,進而搭配微過濾 (microfiltration) 收集富含乳脂肪球膜之分離物;此外,更模擬不同之殺菌條件,進行各種溫度條件 (120℃、90℃與65℃) 於30分鐘下之熱處理,對MFGM機能性之影響。於細胞試驗部分,使用小鼠巨噬細胞株 (RAW 264.7),並分為兩主題探討,包括MFGM對巨噬細胞活化之影響,以及MFGM是否可抑制脂多醣 (lipopolysaccharide;LPS) 所引起之發炎反應。結果顯示,不管是否有經過熱處理之MFGM皆會刺激巨噬細胞分泌TNF-α、IL-1β、IL-6以及IFN-γ,然而120℃熱處理組則具有較低之促發炎細胞激素分泌,可能與其活性膜蛋白質變性,以及顯著較低的細胞存活率有關。此外,各種熱處理之MFGM皆無法降低巨噬細胞因LPS刺激引發之促發炎細胞因子TNF-α、IL-1β及IL-6的分泌,甚至可能有加乘刺激之效果。因此可得知,MFGM分離物可以刺激巨噬細胞活化,並推測與其上之生物活性膜蛋白相關;且MFGM無法降低因LPS引起之發炎反應。   腸炎小鼠模式部分,分成控制組 (control)、DSS對照組、低濃度 (10 mg每日/每隻小鼠) 殺菌MFGM、高濃度 (20 mg每日/每隻小鼠) 殺菌MFGM以及高濃度 (20 mg每日/每隻小鼠) 無殺菌MFGM組;其中殺菌條件皆設為65℃,30分鐘。動物實驗結果顯示,在腸炎預防模型之部分,餵飼MFGM無法減緩因DSS誘發腸炎後所引起小鼠之體重降低、採食量降低、腹瀉、糞便潛血評分以及結腸長度縮短;然而於解剖病理學上,MFGM處理組具有較少損傷評分之趨勢。因此,接下來評估治療腸炎之模型,發現餵飼熱處理之MFGM處理組,皆具有顯著改善腸道發炎後造成之損傷,如具有較長之結腸長度與。 綜上所述,MFGM於細胞試驗具有刺激活化巨噬細胞之能力,但無法降低巨噬細胞之發炎反應,亦無法預防及保護小鼠免受於DSS引起急性腸炎之臨床發炎症狀,不過於治療腸炎之模型中,發現MFGM可減緩因DSS誘發腸炎後引起之臨床症狀與結腸組織損傷,並具有加速腸道修復之功能。
Milk fat globule membrane (MFGM) is a complex biological membrane which coats a triglyceride core and is composed of three layers of phospholipids with mosaic membrane proteins. MFGM is responsible for maintaining the stability and emulsified state of the milk fat in milk. Previous studies have shown that the membrane proteins and phospholipids of MFGM individually elicits many potentials for biological activities, such as cancer prevention, lowering blood cholesterol, promoting phagocytosis to apoptotic cells, promoting the repair and maturation of intestinal epithelium, and immune regulation function. Therefore, the aim of the present study was to better understand the effects of MFGM on macrophage and chemical-induced colitis. In this study, preparation of MFGM enriched isolate from buttermilk was conducted by a novel method using hydroxyapatite (HA) pretreatment followed by microfiltration. In addition, MFGM under different heat treatment at various conditions (120°C, 90°C, and 65°C) was performed at 30 minutes to simulate different processing conditions. In the in vitro experiment, mouse macrophage cell line (RAW 264.7) was used for evaluating the immune-stimulating and anti-inflammatory ability of MFGM. The results showed that, both non-treated and heat-treated MFGM were able to stimulate macrophages to secrete pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IFN-γ. The 120 °C heat-treated group had the lowest secretion of pro-inflammatory cytokines, which might be related to the denaturation of biological MFGM proteins caused by heat or significantly lower cell viability. On the other hand, all heat-treated MFGM could not reduce the secretion of pro-inflammatory cytokines in macrophages stimulated by lipopolysaccharides (LPS). In summary, MFGM isolate is able to stimulate macrophage activation which are presumed to be related to the bioactive membrane proteins in vitro. In opposite, MFGM cannot reduce the inflammatory response in macrophage caused by LPS. In the in vivo mouse colitis model, control group (control), dextran sodium sulfate (DSS) control group (CD), a low concentration (10 mg) pasteurized MFGM (PLMD), a high concentration (20 mg) pasteurized MFGM (PHMD) and high concentration (20 mg) MFGM without pasteurization group (NHMD) were conducted, whereas the condition of pasteurization was 65 ℃, 30 min. The results showed that administration of MFGM could not improve the weight loss, decrease of feed intake, diarrhea, fecal blooding, and colon length shortening caused by DSS-induced colitis in preventing model. In histopathological evaluations, administration of MFGM treated group had a less damage. In conclusion, MFGM has the ability to stimulate the activation of macrophages, but it cannot inhibit the inflammatory response of macrophages, nor be able to prevent mice from DSS-induced colitis. MFGM improves clinical symptoms and colon tissue damage, and has accelerated intestinal repair function after recovery period in healing model.
URI: http://hdl.handle.net/11455/98071
文章公開時間: 10000-01-01
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