酪蛋白糖巨肽对肠粘膜获得性免疫信号通路的作用研究

发布时间：2018-06-15 18:58

本文选题：酪蛋白糖巨肽 + 树突状细胞　；参考：《天津商业大学》2012年硕士论文

【摘要】：酪蛋白糖巨肽(Casein Glycomacropeptide,CGMP)是来源于乳中κ-酪蛋白的一个多肽片断,众多学者研究证实,CGMP具有多种生物活性功能。本研究在以往学者研究的基础上,以购得的进口纯品CGMP为刺激物,研究CGMP对先天免疫和获得性免疫的调节作用。一方面用Balb/c小鼠为实验动物,将一系列不同浓度的CGMP分别以灌胃和腹腔注射两种方式给药,给药2小时后分别眼眶取血,分离血清,利用悬浮液体芯片系统,检测小鼠外周血血清中24种细胞因子的浓度,研究CGMP对小鼠细胞因子网络的影响作用和对小鼠可能产生的先天免疫应答作用。结果口服给药后有显著变化的细胞因子有4种,分别为GM-CSF、IFN-γ、IL-2、MIP-1α;腹腔注射后有显著变化的细胞因子有7种,分别为Eotaxin、TNF-α、G-CSF、GM-CSF、IL-6、IL-17、KC。另一方面以C57BL/6雄性小鼠为实验动物,诱导培养其骨髓源树突状细胞(Dendritic cells,DCs),培养至合适时间后将一系列不同浓度的CGMP添加到DCs培养体系中,分别以空白完全培养基和LPS做为空白对照和阳性对照,继续培养,培养48h后分离细胞培养液和细胞,用流式细胞仪检测DCs的成熟情况;同时,将添加刺激物并培养48h后的DCs与分离纯化后得到的T细胞以不同比例混合,将两种细胞共同孵育96h,用CCK-8试剂盒检测DCs对T细胞的增殖程度的作用。选择DCs对T细胞增殖作用最强烈的两者的比值,同样用CGMP刺激DCs后进行混合淋巴细胞反应。反应结束分离细胞培养上清液,检测其中的24中细胞因子浓度。结果表明:细胞培养上清液中的细胞因子有显著变化的8种分别为G-CSF、GM-CSF、IFN-γ、IL-1α、IL-2、IL-4、IL-5、IP-10;各浓度的CGMP均能促进DCs的成熟,但其促进DCs成熟的作用低于阳性对照LPS;DCs与T细胞的共培养结果显示经CGMP刺激成熟的DCs能促进T细胞增殖,说明DCs对这种肽或其水解的片段具有抗原提呈作用,继而引起相应免疫细胞的活化、增殖。
[Abstract]:Casein glycosylated peptide (CGMPP) is a polypeptide fragment derived from milk 魏 -casein. Many studies have confirmed that CGMP has a variety of bioactive functions. On the basis of previous studies, the effects of CGMP on innate and acquired immunity were studied with imported CGMP as stimulant. On the one hand, Balb / c mice were used as experimental animals. A series of CGMP of different concentrations were given by intragastric and intraperitoneal injection respectively. After 2 hours of administration, blood was taken from the orbit, serum was separated, and the suspension liquid chip system was used. The effects of CGMP on cytokine network and innate immune response in mice were studied by detecting the concentration of 24 cytokines in peripheral blood of mice. Results there were 4 kinds of cytokines which changed significantly after oral administration, namely GM-CSF- 纬 IFN- 纬 IL-2mMIP-1 伪, and 7 kinds of cytokines (eotaxin TNF- 伪 G-CSFU GM-CSFU IL-6IL-17KC) after intraperitoneal injection. On the other hand, C57BL / 6 male mice were used as experimental animals to induce and culture bone marrow-derived dendritic cells (DC) cells, and a series of CGMP of different concentrations were added to DCs culture system. The culture medium and LPS were used as blank control and positive control respectively. After 48 hours of culture, the cell culture medium and cells were separated, and the maturation of DCs was detected by flow cytometry. The DCs cultured for 48 h were mixed with the purified T cells in different proportions. The two kinds of cells were incubated together for 96 h. The effect of DCs on the proliferation of T cells was detected by CCK-8 kit. The ratio of DCs to T cell proliferation was chosen, and the mixed lymphocyte reaction was carried out after DCs were stimulated by CGMP. Cell culture supernatants were isolated at the end of the reaction and 24 of them were detected for cytokine concentration. The results showed that 8 kinds of cytokines in the supernatant of cell culture were G-CSFU GM-CSFU IFN- 纬, IL-1 伪, IL-2mil, IL-4, IL-5, IP-10, and CGMP at different concentrations could promote the maturation of DCs. The results of co-culture of DCs and T cells showed that the mature DCs stimulated by CGMP could promote the proliferation of T cells, indicating that DCs had antigenic effect on this peptide or its hydrolyzed fragments. In turn, it causes the activation and proliferation of the corresponding immune cells.
【学位授予单位】：天津商业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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