維生素對小鼠初代免疫細胞及子代免疫反應的影響

In vitro and In vivo Study on The Immuno-regulatory Effects of Vitamins on Mice

楊政諺(Chen-Yen Yang)

指導教授 ： 林璧鳳

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01808

摘要

已知維生素A視網酸、維生素D 1,25-dihydroxyvitamin D3、維生素E α-toco- pherol與葉酸會參與免疫調控影響免疫反應。本研究分別分離出正常鼠、塵蟎蛋白（Dp2）致敏鼠及口服Dp2致敏鼠的初代脾臟、腸繫膜淋巴結（MLN）和腸道皮耶氏體（PP）細胞，以體外培養模式，比較四種維生素在不同生理情況影響T細胞的效應，並以口服Dp2致敏鼠初代脾臟細胞培養，探討這些維生素對調節型T細胞（Treg）的影響，進一步探究維生素是否能提升口服致敏原所誘發之Treg的表現。最後，以飲食缺乏維生素D之動物模式，探討維生素D對免疫系統的影響。本研究Dp2致敏鼠，利用腹腔注射Dp2致敏原兩次的方式，確實可以成功提高小鼠血清中總IgE抗體的含量，顯示小鼠在兩次腹腔注射之後，已產生過敏免疫反應。口服Dp2致敏鼠除了腹腔注射Dp2之外，並同時連續7天餵食致敏原Dp2。結果顯示，脾臟細胞Treg在Dp2特異性刺激下，Control組與口服組Foxp3+ Treg的百分比均有顯著提升，口服組Treg表現顯著較Control組高，顯示口服Dp2鼠初代細胞在特異性抗原刺激下可以明顯誘發Treg表現。在三項實驗小鼠初代細胞培養模式中，視網酸、1,25-dihydroxyvitamin D3與α-tocopherol皆有減弱IFN-γ或IL-2、增強IL-5細胞激素的作用。1,25-Dihydroxyvitamin D3與α-tocopherol可以促進正常小鼠脾臟細胞分泌IFN-γ。在兩項致敏小鼠脾臟細胞中，視網酸與1,25-dihydroxyvitamin D3均降低抑制性細胞激素IL-10和TGF-β1。葉酸對正常及致敏小鼠脾臟Th1細胞激素沒有影響，但在口服Dp2小鼠細胞顯著增加IFN-γ與IL-2，且明顯提升IL-10和TGF-β1表現。在口服Dp2小鼠腸道淋巴結中，視網酸顯著降低MLN細胞中口服Dp2所誘發之Treg百分比，1,25-dihydroxyvitamin D3則顯著抑制PP細胞Foxp3的表現，只有葉酸明顯增加MLN細胞Treg比例。因此葉酸具有促使口服Dp2鼠Th2傾向之脾臟細胞轉向Th1的作用，也能提高口服致敏原所誘發之Treg的表現，並增加抑制性細胞激素的分泌。本研究餵食親代母鼠缺乏維生素D飼料6週，再使之與公鼠交配產生缺乏維生素D子代小鼠（VDD）。結果顯示，VDD組血清中維生素D營養指標25-hydroxyvitamin D3與肝臟重量均顯著較Control組低。以流式細胞儀分析肝臟、脾臟及腸繫膜淋巴結中各種免疫細胞的組成，發現缺乏維生素D可以顯著改變肝臟中多項免疫細胞的比例，無論6週齡或12週齡VDD組均以肝臟免疫細胞變化最明顯，其中以自然殺手T細胞（Natural Killer T cell, NKT）與B1細胞亞群受維生素D缺乏的影響最顯著。因此，維生素D對於肝臟免疫系統發展成熟的過程扮演不可或缺的角色。

關鍵字

視網酸 ； 1,25-dihydroxyvitamin D3 ； α-tocopherol ； 葉酸 ； 塵蟎蛋白Dp2 ； 口服耐受性 ； Foxp3+調節型T細胞

並列摘要

Vitamin A (retinoic acid), D (1,25-dihydroxyvitamin D3), E (α-tocopherol) and folic acid has been associated with immuno-modulation for a long time. Most of these vitamins involved in the development of Foxp3+ regulatory T cell (Treg). However, it remains unclear whether vitamins are capable of converting already activated and polarized T cells to Tregs and, thereby, to interfere with unwanted, ongoing immune responses. In order to identify the immuno-regulatroy effects of these vitamins on polarized T cells and Tregs, we examined the effects of these vitamins on cytokine production and Treg expression using in vitro primary spleen, mesenteric lymph node (MLN) and Peyer’s patch (PP) cell culture from normal, Der p 2 (Dp2)-sensitized as well as Dp2-sensitized and oral administered BALB/c mice. Treatment with retinoic acid, 1,25-dihydroxyvitamin D3 and α-tocopherol in primary cell culture strongly decreased IFN-γ or IL-2, but enhanced IL-5. Both 1,25-dihydroxyvitamin D3 and α-tocopherol could increase the IFN-γ production in primary splenocyte culture from normal mice. Besides, retinoic acid and 1,25-dihydroxyvitamin D3 strongly inhibited IL-10 and TGF-β1 secretion in splenocyte culture from Dp2-sensitized as well as Dp2-sensitized and oral administered mice. Folic acid had no effects on Th1 cytokine production from normal and Dp2-sensitized mice; however, we found that in primary splenocyte culture from Dp2-sensitized and oral administered mice, folic acid effectively increased IFN-γ and IL-2 production. The inhibitory cytokines, IL-10 and TGF-β1, were also highly produced by splenocytes after in vitro treatment with folic acid. Unlike retinoic acid, folic acid efficiently promote the in vitro generation of Foxp3+ Treg in MLN cell culture from Dp2-sensitized and oral administered mice. These data show that folic acid has the potential of switching Th2-bias T cell response toward Th1 and enhancing the expression of Tregs induced by Dp2 feeding. Furthermore, we investigated the in vivo effects of vitamin D deficiency on phenotypic frequency of immune cells and cytokine secretion in liver, spleen and mesenteric lymph node from vitamin D deficient C57BL/6 mice. We demonstrated that the distribution of immune cells from control mice will be dramatically altered, no matter at age 6-week or 12-week, in vitamin D deficient mice, especially Natural Killer T cell and B1 cell subsets in liver. These data suggest that vitamin D may play an important and necessary role in the normal development of liver immune system.

並列關鍵字

retinoic acid ； 1,25-dihydroxyvitamin D3 ； α-tocopherol ； folic acid ； Der p 2 ； oral tolerance ； Foxp3+ regulatory T cell

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