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標題: | 維生素對小鼠B細胞活性與調節功能之影響 The effects of vitamins on mouse B cells activity and regulatory functions |
作者: | Ya-Chun Yang 楊雅珺 |
指導教授: | 林璧鳳(Bi-Fong Lin) |
關鍵字: | 維生素A,維生素E,B-1細胞,調節型B細胞,IL-10,IL-21, vitamin A,vitamin E,B-1 cells,regulatory B cells,IL-10,IL-21, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 本研究擬探討維生素對小鼠B細胞活性及調節功能的影響,以體外 (in vitro) 細胞培養模式,利用anti-mouse CD45R/B220 magnetic particles 自6 ~ 10週齡BALB/c小鼠之初代脾臟、腹腔及皮耶氏體 (Peyer’s patch) 細胞分離B細胞,亦由腹腔細胞培養而得之B-1細胞,分別以維生素A、D、E及葉酸預處理1小時再以LPS刺激48小時,分析細胞存活率、細胞激素及抗體的分泌。接著以qPCR方法分析小鼠脾臟B細胞中IL-10細胞激素之基因表現,亦以細胞內染分析維生素對調節型B細胞百分比的影響。最後以維生素培養的B細胞與經anti-CD3/anti-CD28活化的CD4+ T細胞共培養,探討經維生素培養的B細胞對T細胞增生能力的影響。
結果顯示,維生素D可抑制脾臟、腹腔B細胞及B-1細胞IL-6、IL-10及IgM的分泌,並抑制皮耶氏體B細胞IL-6及TGF-β1分泌的趨勢,且顯著促進脾臟B細胞IL-10 mRNA的表現;缺乏葉酸或補充10倍葉酸之AIN-76飼料餵食5週的BALB/c小鼠,脾臟B細胞在LPS刺激下,缺乏或補充葉酸皆顯著降低IgM的分泌,且IL-6及IL-10的分泌隨飲食中葉酸的增加而增的趨勢。維生素A可增加LPS刺激下脾臟B細胞IL-6 並抑制IL-10及IgM的分泌,且腹腔B細胞以及B-1細胞結果趨勢也相同,並抑制脾臟B細胞IL-10 mRNA的表現;但是,維生素A可增加脾臟B細胞中CD1dhiCD5+細胞比例,即所謂B10細胞;當IL-21存在時,維生素A可增加脾臟B細胞IL-10之分泌量及IL-10+細胞比例;將與IL-21及維生素A培養的B細胞與CD4+ T細胞共培養後,可顯著抑制T細胞之增生。維生素E可增加腹腔B細胞IL-6、IL-10的分泌,但在脾臟B細胞及B-1細胞中趨勢相反,可促進脾臟B細胞IL-10 mRNA的表現;將與維生素E培養45小時之後的B細胞與CD4+ T細胞共培養後,可顯著促進T細胞增生,若B細胞培養時有IL-21存在,則可顯著抑制T細胞增生。 綜合以上結果,維生素可顯著影響小鼠B細胞亞群的活性,其中維生素A與維生素E在IL-21存在時,可增進B細胞的調節功能,並顯著抑制T細胞的增生。 In order to investigate the effects of vitamins on mouse B cells activity and regulatory function, we isolated B cells from primary splenic, peritoneal and Peyer’s patch cells of 6 to 10-week-old BALB/c mice by using anti-mouse CD45R/B220 magnetic particles, or B-1 cells derived from primary peritoneal cell cultures. B cells were pretreated with vitamin A, D, E or folic acid for 1 hour following LPS stimulation for 48 hours. The cell viability, cytokine and immunoglobulin levels were analyzed. IL-10 cytokine gene expression of splenic B cells was analyzed by qPCR. We also analyzed the effect of vitamin on percentages of regulatory B cells in splenic B cells by intracellular staining, and on the regulatory function of B cells by pretreating B cells with vitamin and then co-culturing with CD4+ T cells for T cell proliferation using 3H-thymidine incorporation assay. The results showed that vitamin D inhibits the IL-6, IL-10, and IgM secretions from spleen and peritoneal B cells, as well as B-1 cells, and also inhibits IL-6 and TGF-β1secretion of Peyer’s patch B cells. Folate supplement had little effects on B-cell subsets activity, only the high-dose folic acid increased spontaneous IL-6 and IL-10 secretion of peritoneal B cells. Folate deficiency tended to increase peritoneal B cells and B-1 cells viability, increase TGF-β1 secretion in splenic, peritoneal and Peyer's patch B cells, and increase IL-10 mRNA expression of splenic B cells. Vitamin A increased IL-6, decreased IL-10, IgM secretions of splenic B cells under LPS stimulation, and the same trend was observed in peritoneal B cells and B-1 cells culture. Vitamin A also decreased IL-10 mRNA expression in spleinc B cells, but increased CD1dhiCD5+ cells percentage in splenic B cells. In the presence of IL-21, vitamin A increased IL-10 secretion and IL-10+ cells percentage in splenic B cells. B cells cultured with IL-21 and vitamin A following co-cultured with CD4+ T cells significantly inhibited T cells proliferation. Vitamin E increased IL-6, IL-10 secretion in peritoneal B cells, but in splenic B cells and B-1 cells showed opposite trend. Vitamin E also increased IL-10 mRNA expression in splenic B cells. B cells cultured with high level of vitamin E for 45 hours can promote T cells proliferation after co-cultured with CD4+ T cells. But if B cells cultured with IL-21 and vitamin E following co-cultured with CD4+ T cells, can significantly inhibit CD4+ T cells proliferation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17312 |
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