本文選題：催乳素　切入點(diǎn)：CD154　出處：《福建醫(yī)科大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】： 目的:為探討催乳素/催乳素受體(PRL/PRLr)介導(dǎo)的免疫應(yīng)答機(jī)制,本課題研究PRL對(duì)免疫應(yīng)答基本過(guò)程的影響并對(duì)信號(hào)傳導(dǎo)途徑進(jìn)行了整體性分析,前者進(jìn)行三方面試驗(yàn),既分別測(cè)試催乳素刺激前后T細(xì)胞活化所需第二信號(hào)共刺激因子CD154和CD28水平、細(xì)胞增殖能力及其免疫效應(yīng)分子IFN-γ/IL-4分泌模式,后者通過(guò)蛋白組學(xué)分析,試圖了解催乳素在免疫細(xì)胞中的信號(hào)網(wǎng)絡(luò)系統(tǒng)。 方法:實(shí)驗(yàn)一應(yīng)用不同劑量的重組人催乳素(rhPRL)和植物血凝素(PHA)單獨(dú)或聯(lián)合刺激人T淋巴白血病細(xì)胞株JurkatD1.1細(xì)胞,利用流式細(xì)胞儀檢測(cè)細(xì)胞表面CD154和CD28分子的表達(dá)量;對(duì)不同時(shí)間點(diǎn)JurkatD1.1細(xì)胞表面CD154和CD28分子的表達(dá)量進(jìn)行測(cè)定。應(yīng)用催乳素受體單克隆抗體(PRLrAb)阻斷催乳素的作用,分別比較JurkatD1.1細(xì)胞表面CD154和CD28分子基礎(chǔ)表達(dá)量和rhPRL聯(lián)合PHA刺激組表達(dá)量在應(yīng)用PRLrAb前后其各自呈現(xiàn)的變化。同時(shí)應(yīng)用半定量RT-PCR測(cè)定對(duì)照組、rhPRL聯(lián)合PHA刺激組和PRLrAb阻斷組JurkatD1.1細(xì)胞表面CD154和CD28分子mRNA的表達(dá)水平。實(shí)驗(yàn)二應(yīng)用臺(tái)盼藍(lán)拒染法通過(guò)倒置顯微鏡進(jìn)行細(xì)胞計(jì)數(shù)以及利用MMT比色法酶標(biāo)儀測(cè)定OD550值來(lái)評(píng)估rhPRL對(duì)JurkatD1.1細(xì)胞增殖的影響。實(shí)驗(yàn)三應(yīng)用酶聯(lián)免疫吸附法(ELISA)檢測(cè)不同濃度rhPRL刺激JurkatD1.1細(xì)胞24小時(shí)后的細(xì)胞上清液中IFN-γ和IL-4水平,并計(jì)算IFN-γ/IL-4比值,籍此來(lái)反映Th1/Th2細(xì)胞因子平衡偏離的方向。實(shí)驗(yàn)四應(yīng)用磷酸化金屬親和層析法(PMAC)富集磷酸化蛋白并用單向凝膠電泳(1DE)分離,同時(shí)利用抗磷酸化酪氨酸抗體免疫沉淀法(IP)富集磷酸化酪氨酸,并用雙向凝膠電泳(2DE)分離。染色后通過(guò)凝膠掃描系統(tǒng)對(duì)不同組的蛋白質(zhì)斑點(diǎn)或條帶進(jìn)行分析,應(yīng)用手工和自動(dòng)挖膠系統(tǒng)獲取差異的蛋白質(zhì)斑點(diǎn)或條帶。酶解后通過(guò)質(zhì)譜分析并與蛋白質(zhì)數(shù)據(jù)庫(kù)進(jìn)行蛋白質(zhì)匹配鑒定,由此鑒定出參與催乳素在JurkatD1.1細(xì)胞內(nèi)信號(hào)傳導(dǎo)的信號(hào)分子。 結(jié)果:單獨(dú)使用重組人催乳素(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- 緯 -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- 緯 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- 緯 / 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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R341

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本文編號(hào)：1623003