【作者】 齐娜；

【导师】 孟爱宏；

【作者基本信息】 河北医科大学 ， 内科学， 2012， 硕士

【摘要】 目的：炎症性肺病（inflammatory lung disease, ILD）是感染、中毒、免疫等各种肺内外因素引起的肺部炎症反应。脂多糖（lipopolysaccharide,LPS）是其重要致病因素。肺泡巨噬细胞（alveolar macrophage, AM）作为肺部第一道防线，在启动和维持炎症反应方面发挥重要作用。LPS作用于AM表面受体，通过细胞内信号级联传递，可释放促炎因子如肿瘤坏死因子（tumor necrosis factor, TNF）-α和抗炎因子如白介素（Interleukin, IL）-10。P38丝裂原活化蛋白激酶（mitogen-activated proteinkinase, MAPK）是参与细胞内信号传导的重要激酶，介导炎症反应，同时信号转导转录激活子-3（signal transducers and activators of transcription-3,STAT3）在LPS导致的炎症瀑布反应中发挥作用[1]。p38MAPK抑制剂SB203580可抑制LPS所致肺部炎症反应。我们假设p38MAPK和STAT3信号通路间存在关联。本实验以小鼠AM为研究对象，观察SB203580对LPS诱导的MH-S细胞上清液IL-10及STAT3活化的影响，探讨p38MAPK与STAT3间关系，为临床治疗ILD提供新的思路。方法：（1） ELISA法测定细胞上清液IL-10浓度：复苏并传代培养MH-S细胞，调节细胞浓度为5×106/ml于24孔板中过夜，随机分组刺激后收集细胞上清液，用ELISA试剂盒检测IL-10浓度。分组：①对照组：加入与实验组等体积的无血清RPMI1640培养液;②LPS组：终浓度100ng/ml LPS;③LPS+SB5组：加入终浓度5μΜ SB203580，20min后加入终浓度100ng/ml LPS;④LPS+SB10组：加入终浓度10μΜSB203580,20min后加入终浓度100ng/ml LPS;⑤LPS+SB15组：加入终浓度15μΜ SB203580，20min后加入终浓度100ng/ml LPS。各组分别刺激90min、2h、4h、6h、12h取细胞上清液，冻存于-80℃，待ELISA检测IL-10浓度。（2）免疫细胞化学法测定STAT3和磷酸化STAT3的表达变化。调整细胞浓度106/ml，于24孔板爬片过夜，按以下分组分别测定STAT3和磷酸化STAT3的表达。分组：①对照组：加入与实验组等体积的无血清RPMI1640培养液;②LPS15min组：终浓度100ng/ml LPS;③LPS15min+SB10组：加入终浓度10μΜ SB203580，20min后加入终浓度100ng/ml LPS。结果：（1）ELISA测定细胞上清液中IL-10含量的变化：LPS刺激MH-S细胞后，细胞上清液中IL-10含量增加，LPS刺激90min，IL-10含量开始升高，6h达最高值，12h含量降低。经单因素方差分析，对照组、LPS组与SB+LPS组，三组间IL-10表达水平有显著性差异（P<0.05）。SB203580（5μΜ、10μΜ和15μΜ）预处理显著抑制LPS诱导的IL-10含量增加，SB203580组无显著量效关系（P＞0.05），IL-10含量仍高于对照组。（2）免疫细胞化学测定STAT3和磷酸化STAT3的表达变化。①STAT3的表达变化：对照组及试验组均细胞质黄染，经单因素方差分析，各组间STAT3表达水平差异无统计学意义（P＞0.05）;②磷酸化STAT3的表达变化：对照组仅见微弱黄染，LPS15min组细胞核黄染增强，LPS15min+SB10组细胞核黄染变淡。经单因素方差分析，两个实验组与对照组比较，磷酸化STAT3表达均显著增高（P<0.05）；与LPS15min组比较，LPS15min+SB10组磷酸化STAT表达显著降低（P<0.05），但与对照组比较LPS15min+SB10组磷酸化STAT3表达仍显著增高（P<0.05）。数据以均数±标准差（x±s）表示。各组用单因素方差分析（One wayANOVA）进行比较。如有显著性用Student-Newman-Keuls（SNK-q）检验，P<0.05表示有统计学意义。结论：1LPS刺激MH-S细胞引起细胞上清中IL-10分泌增加；用p38MAPK抑制剂SB203580干预后，IL-10的分泌减少，仍高于对照组，提示SB203580可能抑制IL-10分泌。随着SB203580剂量增加，IL-10含量差异不显著，提示SB203580剂量增加可能对抗炎细胞因子IL-10无显著性影响。2非活性STAT3存在于细胞浆。LPS刺激MH-S细胞后，STAT3被激活变为有活性的p-STAT3，部分从细胞浆进入细胞核。应用SB203580干预后，细胞核黄染减弱，说明SB203580可能通过p38MAPK减少p-STAT3的表达，提示p38MAPK和STAT3间可能存在关联。3上述结果提示SB203580可能通过p38MAPK抑制STAT3活化，从而抑制IL-10分泌。更多还原

【Abstract】 Objective: Inflammatory lung disease (ILD) is caused by a variety ofintrapulmonary and extrapulmonary factors such as infection, poisoning andimmunodeficiency. Lipopolysaccharide (LPS) is one of the most importantfactors in ILD. The alveolar macrophage (AM) is the first defensive line of thelung and plays an important role in initiation and maintenance of inflammation.LPS activates the receptors of AM, sequently induces the intracellularsignaling cascade and therefore produces a large number of proinflammatorycytokines, such as tumor necrosis factor (TNF)-α, but also of antiinflammatorycytokines, such as interleukin (IL)-10. P38mitogen-activated protein kinase(p38MAPK) is an important kinase involved in intracellular signaltransduction and inflammatory response, and also signal transducers andactivators of transcription-3(STAT3) has been shown to play roles in theinflammatory signaling cascades triggered by LPS. SB203580, p38MAPKspecific inhibitor, can inhibit acute lung injury (ALI) induced by LPS. Weassumed that there was an association between p38MAPK and STAT3signaling pathway. In the study, we observed the influences of SB203580onTNF-α and IL-10production and the activation of STAT3in mouse alveolarmacrophage cell line (MH-S) stimulated by LPS, explored the relationshipbetween p38MAPK and STAT3and this may provide new ideas for clinicaltreatment of ILD.Methods:(1) IL-10production in MH-S cell measured by ELISA. Thecell concentration was adjusted to5×106/ml. Cells were placed in24-wellplates overnight and were stimulated randomly with different agents asfollows:①Control group: with equal volume of RPMI1640of serum-freemedium;②LPS group: LPS stimulation at final concentration of100ng/ml;③LPS+SB5group:5μΜ SB203580was pretreated20mins before100 ng/ml LPS stimulation;④LPS+SB10group:10μΜ SB203580was pretreated20mins before100ng/ml LPS stimulation;⑤LPS+SB15group:15μΜSB203580was pretreated20mins before100ng/ml LPS stimulation. All ofthe supernatant was collected after LPS stimulation for90min,2h,4h,6h,12hand then measured by ELISA.(2) Immunocytochemical determination ofSTAT3and p-STAT3expression. The cell concentration was adjusted to5×106/ml and the cells were stimulated randomly with different agents asfollows:①Control group: with equal volume of RPMI1640of serum-freemedium;②LPS15min group: LPS stimulation at final concentration of100ng/ml for15min;③LPS15min+SB10group:10μΜ SB203580was pretreated20mins before100ng/ml LPS stimulation for15min.The data were expressed as means±SEM. The differences betweengroups were analyzed by one-way analysis of variance (ANOVA). Ifsignificant, the data were analyzed by Student-Newman-Keuls (SNK-q) test.P<0.05was statistically significant.Results:(1) The IL-10levels increased after LPS stimulation, started toincrease at90min, reached peak level at6h, decreased at12h. It wassignificantly different between stimulated group and control group (P<0.05);the levels of IL-10in SB203580(5μΜ,10μΜ and15μΜ) intervention groupdecreased, which were significantly different with LPS group (P<0.05), but itwas not in a concentration dependent manner.(2)①STAT3expressionchanges: cytoplasmic was stained yellow in each group. There was no statistalsignificance between each group (P＞0.05).②p-STAT3expression changes:There was almost no stain in control group. In LPS15min group, the nucleusstained yellow, while LPS15min+SB10group nucleus stained lighter.p-STAT3expression of the two experimental groups inceased significantlycomparing with the control group (P <0.05); Compared with LPS15mingroup, p-STAT3expression decreased significantly in LPS15min+SB10group (P <0.05). While compared with control group, p-STAT3expression ofLPS15min+SB10group increased significantly. Conclusions:1The level of IL-10increased in LPS-stimulated MH-Scell supernatant; the level of IL-10decreased after SB203580intervention,while it was still higher than control group. However the inhibitory effect ofSB203580did not show concentration dependence. It suggested that higherconcentration of SB203580may have no significant impact on the secretion ofthe anti-inflammatory cytokine IL-10.2Non-activated STAT3existed in thecytoplasm. In LPS-stimulated MH-S cells, STAT3was activated into STAT3phosphorylative and entered into the nucleus. After the SB203580intervention,the nuclei stained lighter. It indicated that SB203580may reduce the STAT3phosphorylation expression through p38MAPK and there may be a relationbetween p38MAPK and STAT3signal pathway3. These results suggestedthat SB203580may inhibite STAT3activation through p38MAPK, andtherefore inhibited the secretion of IL-10.更多还原