催乳素免疫调节效应的研究及其信号传导途径的蛋白质组分析

发布时间：2018-03-17 03:32

本文选题：催乳素　切入点：CD154　出处：《福建医科大学》2006年博士论文　论文类型：学位论文

【摘要】： 目的:为探讨催乳素/催乳素受体(PRL/PRLr)介导的免疫应答机制,本课题研究PRL对免疫应答基本过程的影响并对信号传导途径进行了整体性分析,前者进行三方面试验,既分别测试催乳素刺激前后T细胞活化所需第二信号共刺激因子CD154和CD28水平、细胞增殖能力及其免疫效应分子IFN-γ/IL-4分泌模式,后者通过蛋白组学分析,试图了解催乳素在免疫细胞中的信号网络系统。方法:实验一应用不同剂量的重组人催乳素(rhPRL)和植物血凝素(PHA)单独或联合刺激人T淋巴白血病细胞株JurkatD1.1细胞,利用流式细胞仪检测细胞表面CD154和CD28分子的表达量;对不同时间点JurkatD1.1细胞表面CD154和CD28分子的表达量进行测定。应用催乳素受体单克隆抗体(PRLrAb)阻断催乳素的作用,分别比较JurkatD1.1细胞表面CD154和CD28分子基础表达量和rhPRL联合PHA刺激组表达量在应用PRLrAb前后其各自呈现的变化。同时应用半定量RT-PCR测定对照组、rhPRL联合PHA刺激组和PRLrAb阻断组JurkatD1.1细胞表面CD154和CD28分子mRNA的表达水平。实验二应用台盼蓝拒染法通过倒置显微镜进行细胞计数以及利用MMT比色法酶标仪测定OD550值来评估rhPRL对JurkatD1.1细胞增殖的影响。实验三应用酶联免疫吸附法(ELISA)检测不同浓度rhPRL刺激JurkatD1.1细胞24小时后的细胞上清液中IFN-γ和IL-4水平,并计算IFN-γ/IL-4比值,籍此来反映Th1/Th2细胞因子平衡偏离的方向。实验四应用磷酸化金属亲和层析法(PMAC)富集磷酸化蛋白并用单向凝胶电泳(1DE)分离,同时利用抗磷酸化酪氨酸抗体免疫沉淀法(IP)富集磷酸化酪氨酸,并用双向凝胶电泳(2DE)分离。染色后通过凝胶扫描系统对不同组的蛋白质斑点或条带进行分析,应用手工和自动挖胶系统获取差异的蛋白质斑点或条带。酶解后通过质谱分析并与蛋白质数据库进行蛋白质匹配鉴定,由此鉴定出参与催乳素在JurkatD1.1细胞内信号传导的信号分子。结果:单独使用重组人催乳素(rhPRL)或者植物血凝素(PHA)并不能刺激
[Abstract]:Aim: to investigate the mechanism of PRL / PRLr-mediated immune response mediated by prolactin / prolactin receptor (PRL / PRLr), we studied the effect of PRL on the basic process of immune response and conducted a holistic analysis of the signal transduction pathway. Before and after prolactin stimulation, the levels of the second signal costimulator CD154 and CD28, the cell proliferation ability and the secretion pattern of IFN- 纬 -IL-4, which were analyzed by proteomics, were measured, respectively. An attempt was made to understand the signaling network system of prolactin in immune cells. Methods: different doses of recombinant human prolactin prolactin (rhPRL) and phytohemagglutinin (PHA) were used to stimulate human T-lymphoblastic leukemia cell line JurkatD1.1 cells alone or in combination. Flow cytometry was used to detect the expression of CD154 and CD28 on the cell surface. The expression of CD154 and CD28 molecules on the surface of JurkatD1.1 cells at different time points were measured. The prolactin receptor monoclonal antibody (PRL rAb) was used to block the prolactin effect. The basic expression of CD154 and CD28 on the surface of JurkatD1.1 cells and the expression of rhPRL combined with PHA were compared before and after PRLrAb treatment. Meanwhile, the semi-quantitative RT-PCR was used to detect the changes of CD154 and CD28 in the control group combined with PHA stimulation group and PRLrAb blocking group. The expression level of CD154 and CD28 molecules mRNA on the surface of JurkatD1.1 cells. In experiment 2, the effect of rhPRL on the proliferation of JurkatD1.1 cells was evaluated by using trypan blue exclusion method to count cells by inverted microscope and OD550 value measured by MMT colorimetric assay. In experiment 3, the levels of IFN- 纬 and IL-4 in the supernatant of JurkatD1.1 cells stimulated with different concentrations of rhPRL for 24 hours were detected by Elisa. The ratio of IFN- 纬 / IL-4 was calculated to reflect the deviation of Th1/Th2 cytokine balance. In experiment 4, phosphorylated metal affinity chromatography (PMAC) was used to enrich phosphorylated protein and separate it by unidirectional gel electrophoresis. At the same time, the phosphorylated tyrosine was enriched by the immunoprecipitation method of anti-phosphorylation tyrosine antibody (IPP) and separated by two-dimensional gel electrophoresis (2-DE). After staining, the protein spots or bands of different groups were analyzed by gel scanning system. The differential protein spots or bands were obtained by manual and automatic gumming system. The signal molecules involved in the signal transduction of prolactin in JurkatD1.1 cells were identified by mass spectrometry analysis and protein matching identification with protein database. Results: recombinant human prolactin (rhPRL) or phytohemagglutinin (PHA) alone could not be stimulated.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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