【作者】 王大勇；

【导师】 于庆海； 锅岛俊隆；

【作者基本信息】 沈阳药科大学 ， 药理学， 2003， 博士

【摘要】 本研究的目的是为了深入探讨合成鱼腥草素的免疫调节作用。在广泛查阅有关合成鱼腥草素免疫调节作用资料的基础上，本研究首先考察合成鱼腥草素对巨噬细胞功能及活化状态的影响。在获得了一些研究发现的基础上，进一步考察了合成鱼腥草素在巨噬细胞内的细胞信号传导途径，并且增加了与合成鱼腥草素的免疫佐剂作用有关的研究内容。 为了达到最初的实验目的，第一章研究合成鱼腥草素对巨噬细胞功能和活化状态的影响。在这一章中，首先，采用溶菌法考察合成鱼腥草素对巨噬细胞分泌溶菌酶水平的影响；其次，采用游离酚法研究合成鱼腥草素对巨噬细胞产生的耐酒石酸酸性磷酸酶水平的影响；第三，采用显微镜观察法考察合成鱼腥草素对巨噬细胞吞噬功能的影响；第四，采用溶血法测定合成鱼腥草素对巨噬细胞产生的补体C4水平的影响；第五，采用酵母花环法检测合成鱼腥草素对巨噬细胞表面C3b受体水平的影响；第六，采用流式细胞术法观察合成鱼腥草素对巨噬细胞呼吸爆发的影响；第七，采用ELISA法探讨合成鱼腥草素对巨噬细胞产生IL-1α及IL-1β水平的影响。 在第一章已经取得的研究结果的基础上，第二章进一步考察了合成鱼腥草素在巨噬细胞内的信号传导途径。首先，采用荧光分光光度法研究合成鱼腥草素对巨噬细胞内游离钙离子浓度的影响；其次，采用Western-blotting法分析合成鱼腥草素对巨噬细胞内CaMKⅡ、CREB和ERK 1/2磷酸化程度以及Fos蛋白表达水平的影响。 在第一章实验中发现合成鱼腥草素能够促进巨噬细胞产生IL-1β。对于T淋巴细胞激活和增殖具有重要作用的细胞因子，一种是巨噬细胞产生的T细胞协同激活因子IL-1，另一种是T淋巴细胞产生的IL-2。为了进一步探讨并验证合成鱼腥草素的免疫调节作用，在第三章实验中，首先，采用MTT法测定合成鱼腥草素对脾细胞增殖的影响；其次，采用胸腺细胞增殖法检测合成鱼腥草素对脾细胞产生的IL-2水平的影响；第三，采用ELISA法测定合成鱼腥草素对纯化外周血T淋巴细胞产生IL-2水平的影响。 第四章是合成鱼腥草素免疫佐剂作用研究。在这一章中，首先，采用溶血法测定合成鱼腥草素连续给药后对绵羊红细胞抗体IgG和IgM产生的影响；其次，采用ELISA法分析和评价合成鱼腥草素的典型免疫佐剂作用。 在第一章有关巨噬细胞功能和活化状态的研究中发现，合成鱼腥草素能够提高巨噬细胞产生的溶菌酶、酸性磷酸酶、补体 C4以及 IL刁 p水平，增强巨噬细胞的吞噬功能，促进巨噬细胞呼吸爆发，但对巨噬细胞表面C3b受体以及巨噬细胞产生的IL－la水平没有明显影响。溶菌酶、酸性磷酸酶、IL一 水平上升以及呼吸爆发现象都是巨噬细胞活化标志。巨噬细胞的呼吸爆发可以由吞噬过程引起，但吞噬过程并不一定伴有呼吸爆发，例如由C3b受体介导的吞噬过程即不伴有呼吸爆发。根据已有报道，巨噬细胞的呼吸爆发需要细胞内钙离于信号参与。在第二章的合成鱼腥草素在巨噬细胞内信号传导途径研究中发现，合成鱼腥草素能够提高巨噬细胞内游离钙离子浓度，促进蛋白激酶 CaMK 11以及细胞核活化转录因子 CREB磷酸化，从而使它们进人活化状态。合成鱼腥草素还能够提高巨噬细胞内即早基因C－fOS表达产物F。S蛋白水平。根据合成鱼腥草素在巨噬细胞内信号传导途径的研究结果，可以认为合成鱼腥草素首先提高巨噬细胞内游离钙离子浓度，钙离子浓度升高以后会引起 CaMK 11的自身磷酸化，磷酸化 CaMK 11进一步使转录因子 CREB磷酸化，磷酸化 CREB进入细胞核诱导 C－fos基因以及其它基因的表达，c－fos基因表达产物F。S是激活因子蛋白（AP－1）的可变组分，可以进一步调节其它基因表达。 在第三章的研究中发现，合成鱼腥草素能够促进脾细胞增殖，提高体外培养脾细胞分泌的IL－2水平，以及提高纯化外用血T淋巴细胞产生的IL－2水平。 第四章发现，合成鱼腥草素能够促进绵羊红细胞抗体IgG和 IgM的产生。另外还发现，合成鱼腥草素具有典型的兔疫性剂作用。 以上研究结果提示，合成鱼腥草素具有免疫调节作用和免疫佐剂作用。合成鱼腥草素促进巨噬细胞活化，增强巨噬细胞功能，提高巨噬细胞产生的 IL上 及 T淋巴细胞产生的fi一二水平等免疫调节作用可能是其免疫佐剂作用的药理学作用机理的一部分。更多还原

【Abstract】 The purpose of this research was to investigate the immunoregulation activities of sodium houttuyfonate. On the basis of searching the literatures about the immunoregulative activities of sodium houttuyfonate broadly, we first focused our researches on the functions and activation state of macrophages. After making some discoveries, we further investigated the signal transduction pathways of sodium houttuyfonate in macrophages, and some researches related with the adjuvanticity of sodium houttuyfonate.In order to reach our initial aim, the functions and activation state of macrophages were studied in the first section of this research. In the first section, the effect of sodium houttuyfonate on the level of lysozyme secreted by macrophages was first tested by bacteriolytic method; then the effect of sodium houttuyfonate on the level of tartaric acid resistant acid phosphatase produced in macrophages was investigated by analyzing the phenol generated in the reaction that was catalyzed by the enzyme; the third, the effect of sodium houttuyfonate on the phagocytic activity of macrophages was observed by microscopy; the forth, the effect of sodium houttuyfonate on the production of complement C4 from macrophages was tested by hemolytic method; the fifth, the effect of sodium houttuyfonate on the expression of C3b receptors on the surface of macrophages was analyzed by observing the formation percentage of zymosan wreath; the sixth, the effect of sodium houttuyfonate on the respiratory burst in macrophages was tested by flow cytometry; the seventh, the effect of sodium houttuyfonate on the production of IL-la and IL-lp from macrophages was tested by ELISA method.In the second section, the signal transduction pathways of sodium houttuyfonate in macrophages were investigated. First, the changes of the concentration of free calcium ion in macrophages caused by sodium houttuyfonate was analyzed by spectrofluorophotometry; second, the effects of sodium houttuyfonate on the phosphoralation of CaMK II, CREB and ERK 1/2, and the expression of Fos were analyzed by Western-blotting method.It was discovered in the first section that the production of IL-1 was promoted by sodium houttuyfonate treatment. As we know, there are two kinds of cytokines that are most important for the activation and proliferation of T cells, one is the IL-1 generated by macrophages which is the co-stimulator for the activation of T cells, the other is the IL-2 produced by activated T cells themselves, hi the third section, in order to further investigate the immnoregulation activities of sodium houttuyfonate, the action of sodium houttuyfonate on the proliferation of cells in spleen was tested by MTT spectrophotometry;second, the activity of sodium houttuyfonate on the production of IL-2 from the cells in spleen was investigated by measuring the proliferation of thymocytes promoted by IL-2 secreted by the cells in spleen; the third, the action of sodium houttuyfonate on the production of IL-2 from the purified T cells in peripheral blood was measured by ELISA method.The researches in the forth section dealt with the adjuvanticity of sodium houttuyfonate. First, the effects of consecutive medication of sodium houttuyfonate on the generation of anti-sheep serum blood cell antibodies IgG and IgM were tested by hemolytic method; second, the typical adjuvanticity of sodium houttuyfonate was analyzed by ELISA method.It was found in the first section that the production of lysozyme, tartaric acid resistant acid phosphatase, complement C4 and IL-10, the phagocytic function of macrophages, and the respiratory burst in macrophages were enhanced or promoted by sodium houttuyfonate, however, the expression of C3b receptors on the surface of macrophages and the production of IL-1a from macrophages were not significantly affected. The increase of the production of lysozyme, acid phosphatase and IL-1, and the respiratory burst are all the signs of the activation of macrophages. The respiratory burst may be caused by phagocytosis, however, the ph更多还原