【作者】 冯俊涛；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 研究背景白血病抑制因子(leukemia inhibitory factor，LIF)具有神经营养功能，对神经元的生存、分化和功能成熟起到促进作用，能够促使神经元产生神经激肽受体-1(neurokinin receptor 1，NK-1R)等速激肽类相关物质。研究表明，哮喘患者及哮喘模型动物体内LIF表达水平显著高于相应的对照群体，LIF可能参与哮喘等气道炎症性疾病的发病过程，并且发现哮喘患者气道NK-1R表达的上调可能系体内LIF增加所致。作为细胞因子，LIF必须依赖于信号转导通路的介导才能发挥其生物学作用。JAK／STAT和MAPK信号转导通路与LIF的生物学效应密切关联，而这些通路已被证实参与哮喘气道炎症的诸多病理生理过程。支气管上皮细胞作为气道的重要保护屏障，哮喘等多种气道炎症性疾病的发生与支气管上皮的受损存在直接关系。我们的前期研究发现，LIF和NK-1R在哮喘大鼠支气管肺组织中表达均显著高于对照组，支气管上皮细胞是其主要阳性反应细胞。因此，本研究拟以正常人支气管上皮细胞作为靶细胞，探讨LIF通过调节NK-1R等的变化参与气道神经源性炎症形成过程中的信号转导机制。第一章LIF通过STAT和MAPK信号转导通路上调哮喘大鼠肺组织中NK-1R的表达目的观察抗LIF干预对哮喘大鼠支气管肺组织中NK-1R表达的影响，以及在此过程中p-STAT3和p-ERK1／2的表达变化情况，以期探讨LIF参与气道炎症形成过程中的信号转导机制及其与NK-1R之间的调控关系。方法30只SD大鼠随机均分为3组，每组10只(正常对照组，哮喘模型组，LIF抗体干预组)。用OVA致敏、激发方法建立哮喘模型，对哮喘大鼠施以LIF抗体。干预完成后，取各组大鼠肺组织。观察各组大鼠支气管肺组织病理学变化，用免疫组化方法检测各组大鼠支气管肺组织中LIF、p-STAT3、p-ERK1／2及NK-1R的蛋白表达情况。结果哮喘模型组大鼠气道周围有大量炎症细胞浸润，支气管壁和肺泡间隔显著增厚；而与之相比，LIF抗体干预组大鼠气道周围炎症细胞浸润程度明显减轻，支气管壁和肺泡间隔增厚亦有所改善。免疫组化结果提示，哮喘组大鼠气道上皮及其周围组织可见大量的NK-1R、LIF、p-STAT3及p-ERK1／2蛋白阳性细胞，其阳性产物的平均灰度值均明显低于正常对照组(灰度值越低提示表达量越高，反之亦然)，而抗LIF干预组大鼠气道上皮及其周围组织中NK-1R、LIF、p-STAT3及p-ERK1／2蛋白阳性细胞较哮喘组明显减少，呈中等偏弱的表达强度，其阳性产物的平均灰度值均明显高于哮喘组。结论哮喘大鼠气道炎症形成过程中其支气管肺组织中NK-1R的表达水平可能受到LIF的调控，而LIF的作用可能通过JAK／STAT和MAPK信号转导通路的介导得以实现。第二章LIF通过STAT和MAPK信号转导通路上调NHBE细胞中NK-1R的表达目的观察JAK／STAT和MAPK信号转导通路的抑制剂及激动剂对LIF诱导的NHBE细胞中NK-1R的表达的影响，进一步探讨LIF通过调节NK-1R的变化参与气道神经源性炎症形成过程中的信号转导机制。方法培养正常人支气管上皮细胞(NHBE细胞)，用LIF对NHBE细胞进行刺激，在此过程中分别用AG490(JAK2抑制剂)、PD98059(ERK1／2抑制剂)、PMA(蛋白激酶C激动剂)及STAT3-siRNA对NHBE细胞进行预处理。采用Western-blot、RT-PCR及免疫细胞化学方法对上述各干预组细胞进行检测，观察NK-1R、p-STAT3、total-STAT3、p-ERK1／2及total-ERK1／2等指标的表达变化情况。结果LIF能够诱导NHBE细胞中NK-1R、p-STAT3和p-ERK1／2的表达，而total-STAT3和total-ERK1／2的表达水平在LIF刺激前后无明显变化。进一步发现，LIF对NK-1R的诱导表达作用能够分别被AG490和PD98059所抑制；AG490能够抑制LIF对p-STAT3的诱导表达作用，而PD98059对p-STAT3的表达水平无明显影响；反过来，PD98059能够抑制LIF对p-ERK1／2的诱导表达作用，而AG490对p-ERK1／2的表达水平亦无明显影响。PMA能够诱导NHBE细胞中p-ERK1／2和NK-1R表达增加，但未发现PMA能够增强LIF的诱导效应。STAT3-siRNA能够特异性地抑制LIF对p-STAT3的诱导作用，但对p-ERK1／2的表达水平无明显影响；进一步发现，STAT3-siRNA能够抑制LIF对NK-1R的诱导表达作用。结论LIF能够上调NHBE细胞中NK-1R的表达，LIF的上述作用可能是通过JAK／STAT和MAPK信号转导通路的介导得以实现；在LIF上调NHBE细胞中NK-1R表达的过程中，本研究未发现JAK／STAT和MAPK两个信号转导通路之间存在交叉作用。更多还原

【Abstract】 Background: leukemia inhibitory factor(LIF) has been implicated invarious processes of neuronal development, differentiation, survival andneurogenesis, and it was indicated that LIF could increase the expressionof substance P and its receptor(neurokinin-1 receptor, NK-1R). Highlevels of LIF were found in atopic patients and asthmatic rats, and LIFmay be an important signal molecule in the airway response toinflammation. As a kind of cytokines, the biological effects of LIFdepend on signal transduction pathway such as janus kinase/signaltransducer and activator of transcription (JAK/STAT) pathway and theras-mitogen activated protein kinase (MAPK) pathway. It is indicated thatboth JAK/STAT pathway and MAPK pathway are related to asthma.Bronchial epithelial cell is a barrier to airway structure, and it is animportant target cell type in most respiratory diseases such as asthma.High levels of LIF and NK-1R were observed in bronchial epithelial cellsof asthmatic rats, so the bronchial epithelial cell is considered as thetarget cell to explore the signal transduction mechanism of NK-1Rregulation by LIF in this study.Chapter One LIF up-regulates NK-1R expression in lungs ofasthmatic rats via JAK/STAT pathway and MAPK pathway.Objective: To explore the regulation mechanism between LIF andNK-1R in asthma through comparing the expression of NK-1R, LIF,p-STAT3 and p-ERK1/2 between asthmatic rats and asthmatic rats treatedwith anti-LIF.Methods: 30 Sprague-Dawley rats were divided into 3 groups atrandom(asthmatic group, anti-LIF group and control group, n=10), andthey were housed under specific pathogen-free conditions. Sensitizationand provocation(the asthmatic group and the anti-LIF group) wereproduced with OVA, in this process, the anti-LIF group were treated withanti-LIF. Then the rats were killed and lung tissues were fixed in 4%polyoxymethylene, embedded in paraffin, and finally sliced into sections. Immunoreactivity for p-STAT3, p-ERK1/2, NK-1R and LIF proteins weredetected in the lungs of these rats.Results: Compared with the control, inflammatory cell infiltration werefound around airway in asthmatic rats, in addition, alveolar septum andbronch-wall were thickened in asthmatic rats. Compared with theasthmatic, the extent of inflammatory cells infiltration was relieved in therats treated with anti-LIF, and the pathological changes of bronch-wallwere also improved. Immunohistochemistry indicated a higher expressionof LIF in the asthmatic rats compared to that in the control group.Consistent with that, similar changes were observed for NK-1R, p-STAT3and p-ERK1/2. The main positive cell type was airway epithelial cell, andother positive types were also observed such as lymphocyte.Conclusions: NK-1R expression in lungs of asthmatic rats may beregulated by LIF, and it is possible that the effect of LIF is mediated byJAK/STAT pathway and MAPK pathway.Chapter Two LIF up-regulates NK-1R expression in NHBE cellsvia JAK/STAT pathway and MAPK pathway.Objective: To investigate whether LIF induces the expression of NK-1Rin bronchial epithelial cells and whether JAK/STAT pathway andMAPK/ERK pathway participate in this process.Methods: we treated normal human bronchial epithelial (NHBE) cellswith LIF in the presence or absence of AG490(JAK2 inhibitor),PD98059(ERK inhibitor), PMA(the activator of protein kinase C) and thesiRNA against STAT3. Then the expression of NK-1R, p-STAT3,total-STAT3, p-ERK1/2 and total-ERK1/2 in NHBE cells were detectedby Western-blot, RT-PCR or immunocytochemistry.Results: LIF induced activation of p-STAT3, and p-STAT3 expressionwas inhibited by AG-490 but not by PD-98059. Nevertheless, theexpression of total-STAT3 was not affected by the factors mentionedabove. LIF induced activation of p-ERK1/2, and p-ERK1/2 was inhibited by PD-98059 but not by AG-490. Similar to total-STAT3, the expressionof total-ERK1/2 did not change. PMA increased the expression ofp-ERK1/2 in NHBE cells, but there were no significant differencebetween the cells stimulated with LIF and the cells stimulated with LIF inthe presence of PMA. LIF induced expression of NK-1R in NHBE cells,both AG-490 and PD-98059 suppressed the LIF-induced expression ofNK-1R; on the contrary, PMA increased the expression of NK-1R inNHBE cells. LIF-induced p-STAT3 was inhibited by STAT3-siRNA, inaddition, the expression of total-STAT3 was also inhibited bySTAT3-siRNA. However, STAT3-siRNA did not affect the expression ofp-ERK1/2 and total-ERK1/2. The LIF-induced expression of NK-1R wasinhibited by STAT3-siRNA both at the mRNA level and the pretein level,but it was not affected by negative control siRNA and sham plasmid.Conclusions: LIF up-regulates NK-1R expression in NHBE cells viaJAK2/STAT3 pathway and MAPK pathway, in this process, noobservable interaction was found between the two pathways.更多还原