【作者】 汪诚；

【导师】 刘荣玉；

【作者基本信息】 安徽医科大学 ， 老年医学， 2007， 博士

【摘要】 研究背景近年来,嗜酸性粒细胞被认为是哮喘发病机制中的主要效应细胞。由于嗜酸性粒细胞侵入到支气管粘膜以及随后所释放的大量的毒性物质,因此常常引起支气管上皮细胞的损害,从而引起支气管的高反应性。另一方面,支气管上皮细胞也有吞噬凋亡的嗜酸性粒细胞的能力,这对炎症的消散有着重要的意义。这个过程受着许多因素的调节,这些调节因素可能有着重要的临床治疗意义。膜连蛋白Ⅰ是13个结构相关的钙依赖的膜连接蛋白Annexin超家族成员之一。Fan等研究发现膜联蛋白Ⅰ作为配体和受体促进巨噬细胞吞噬人外周血的淋巴细胞。另外,膜联蛋白Ⅰ存在于II型肺泡细胞系A549细胞的表面,地塞米松增加A549细胞表面膜联蛋白Ⅰ的表达,由此我们推测膜联蛋白Ⅰ可能参与地塞米松上调A549细胞吞噬凋亡的嗜酸性粒细胞的过程。目的在这次研究中,我们观察了A549细胞吞噬凋亡的嗜酸性粒细胞的动态过程,并且探讨了膜连蛋白I是否参与地塞米松上调A549细胞吞噬凋亡的嗜酸性粒细胞的过程,以及凋亡的嗜酸性粒细胞的吞噬对A549细胞分泌IL-6和IL-8的影响。方法1.嗜酸性粒细胞的分离和凋亡用红细胞裂解液去除红细胞后,利用磁性活化的细胞分离柱和CD15、CD16的磁珠抗体进行分离人外周血的嗜酸性粒细胞。纯化的嗜酸性粒细胞在培养箱中培养48h进行老化凋亡,然后用膜连蛋白V-FITC凋亡试剂盒进行凋亡评估。2. A549细胞吞噬凋亡的嗜酸性粒细胞的观察将A549细胞放入到热台系统中,显微镜下调整好界面。此时将凋亡的嗜酸性粒细胞从小孔中加入到35mm培养皿中,每隔一分钟自动拍摄吞噬过程。对吞噬的A549细胞在MetaMorph图像分析软件中进行周长、面积和密度的测量。GFP转染的A549吞噬7-AAD标记的凋亡的嗜酸性粒细胞用激光共聚焦拍摄。3.A549细胞吞噬凋亡的嗜酸性粒细胞的定量观察吞噬试验前,把长满A549细胞的玻片转移到用Parafilm膜包被好的12孔板中。把凋亡的嗜酸性粒细胞悬浮到DMEM中,加入到玻片上(1×104细胞/板),在37°C中孵育60分钟。吞噬结束后,用EDTA-PBS清洗以除去未吞噬的凋亡的嗜酸性粒细胞后,接着用4%的多聚甲醛固定,o-dianisidine进行染色,进行计数。3.膜连蛋白I的Western blot分析为了提供足够的A549细胞以测量A549细胞表面的膜连蛋白I,A549细胞接种于100mm的培养皿中培养96h,弃去培养基,用EDTA(1mmol/ml)洗A549细胞,收集上清蛋白,接着进行Western Blot分析。4.细胞因子的测定用地塞米松处理或未处理的A549细胞在48孔板中单独孵育或与凋亡的嗜酸性粒细胞共同孵育,收集上清。用IL-6和IL-8放射免疫试剂盒分析上清中的IL-6和IL-8水平。TGF-β1由ELISA试剂盒测定。5.统计方法所有试验分5次独立完成,数据用均数±标准误表示。组间的差异用单因素的方差分析,而两组之间的差异用t检验,统计在SPSS10.0软件统计包中进行,P<0.05表示差异有显著性。结果1.动态观察A549细胞吞噬凋亡的嗜酸性粒细胞自动地拍摄A549细胞吞噬凋亡的嗜酸性粒细胞的动态的过程。对吞噬前30分钟、吞噬时、吞噬后60分钟的A549细胞的周长、面积和密度进行测量发现,三个观察的指标在吞噬前30分钟、吞噬时、吞噬后60分钟差异有显著性,而在吞噬时和吞噬后60分钟三个观察指标并无明显的改变。激光共聚焦也证实了吞噬现象。2.地塞米松促进A549细胞吞噬凋亡的嗜酸性粒细胞在不同的时间内预先加入地塞米松后,A549细胞吞噬能力的发生了变化。在地塞米松孵育1小时开始吞噬功能增加,4小时达到最高峰为55%±2.2% ,而到24小时又降到38%±1.4%,但都显著高于对照组。3.地塞米松增加A549细胞表面膜连蛋白I的表达蛋白质印迹试验分析了地塞米松增加A549细胞表面膜连蛋白I的表达的作用。其密度值在2-4小时为最强,24小时降至正常范围,显示出地塞米松以时间依赖的方式增加A549细胞表面膜连蛋白I的表达。4.膜连蛋白I的单克隆抗体抑制地塞米松对A549细胞吞噬凋亡的嗜酸性粒细胞的增加作用为了探讨A549细胞表面膜连蛋白I对吞噬功能的影响,我们把膜连蛋白I单克隆抗体(10μg/ml)加入到经地塞米松在不同时间处理的A549细胞中进行孵育(30 min at 37°C),结果显示经地塞米松和单克隆抗体同时处理的A549细胞的吞噬能力明显地低于单用地塞米松处理的A549细胞的吞噬能力,差异有显著性。5.凋亡的嗜酸性粒细胞的表面膜连蛋白I参与了A549细胞的吞噬过程凋亡的嗜酸性粒细胞用EDTA洗以除去膜表面的膜连蛋白I,然后加入到A549细胞中,结果表明抑制了A549细胞的吞噬能力,与不处理组比较差异有显著性。6.地塞米松增加的A549细胞吞噬并不激发A549细胞分泌炎症因子的作用凋亡的嗜酸性粒细胞的吞噬和地塞米松对吞噬能力的增加并不能激发IL-6 IL-8和TGF-β1的释放。结论1.A549细胞在吞噬凋亡的嗜酸性粒细胞后其周长、面积和密度发生了变化,周长和面积缩小,而密度增加。2.地塞米松通过膜连蛋白I来增加A549细胞吞噬凋亡的嗜酸性粒细胞的能力。3.地塞米松增加的A549细胞吞噬并不激发A549细胞分泌炎症因子的作用。更多还原

【Abstract】 BackgroundEosinophils are recently confirmed as one of the major effector cells in the immuno-inflammation changes in the airway in asthma. By infiltrating into the bronchial mucosa and subsequent release of histotoxic substances, eosinophils may cause epithelial damage, an event thought to be important in the development of airway hyperresponsiveness. On the other hand, the bronchial epithelial cells have the ability to engulf the apoptotic eosinophils, which may be crucial to the solution of inflammation. The process is regulated by many factors and the regulators of this process may have therapeutically potential.Annexin I is a member of annexin superfamily of 13 structurally related proteins that are able to bind to cell membrane in a calcium-dependent manner. Interestingly Annexin I may also be an endogenous ligand mediating the engulfment of apoptotic cells. Fan et al found that annexin I served as both ligand and receptor in promoting the phagocytosis of Jurkat T lymphocytes and human peripheral T lymphocytes by macrophages. In addition, annexin I is expressed on the membrane surface of A549 cell and is up-regulated by dexamethasone, indicating that annexin I may be involved in the dexamethasone’s upregulation of the engulfment of apoptotic eosinophils in A549 cell.ObjectiveIn the present study, we observed the real time process of phagocytosis of apoptotic eosinophils by A549 cells and investigated whether Annexin I was involved in the dexamethasone-mediated upregulation of phagocytosis of apoptotic eosinophils by A549 cells. In the meanwhile, we investigated the effect of phagocytosis of apoptotic eosinophils on IL-6 and IL-8 release in A549 cells. Method1. Eosinophil isolation and apoptosis inductionAfter removal of red blood cells using red blood cell lysis buffer, the eosinophils were isolated by micromagnetic beads coated with anti-CD16, anti-CD15 and the magneticactivated separation column. Flow cytometric analysis assessed the apoptosis eosinophils rate after culturing for 48 hours.2. Real time process of phagocytosis of apoptotic eosionphils by A549 cells.A549 Cells were put into the heated-plate system. After this, apoptotic eosinophils were put into the dishes through the hole. The whole process of phagocytosis was automatically recorded every one minute. The curve length, area and density of A549 cells before and after phagocytosis were measured by MetaMorph software.A549 cells transfected by GFP engulf apoptotic eosinophils labled with 7-AAD, which was scanned by laser confocal scanning microscope.3. Quantitative observation of phagocytosis of apoptotic eosionphils by A549 cells.The coverslip with A549 cells were transferred to new 12-well plates coated with Parafilm membrane one by one. Washed apoptotic eosinophils were resuspended in DMEM and added to the coverslip (1×104/slide) with A549 cells for 60 minutes at 37°C under 5%CO2. After interaction, the noningested eosinophils were washed using oscillator with Ca2+ and Mg2+ free PBS solution supplemented with EDTA before fixation. The ingested eosinophils were visualized by o-dianisidine staining. At least 200 A549 cells which were conter-stained with haematoxylin in duplicate wells in randomly selected fields were counted by a blinded investigator, and the proportion of ingested eosinophils was expressed as a percentage.4. Western blotting analysis of annexin IIn order to provide a sufficient number of cells for measurement of cell surface annexin I induction, subconfluent A549 cells were seeded in DMEM medium in 100mm dishes. The supernatants washed with EDTA were collected and followed by western blot analysis.5. Cytokine productionA549 cells unstimulated or stimulated with dexamethasone (10-6 M) were incubated in DMEM in 48 wells plates alone or togather with apoptotic eosinophils. IL-6 and IL-8 were assayed in supernatants from cocultures of A549 cells and apoptotic eosinophils by RIA kit according to the manufacturer’s instruction. Concentration of TGF-β1 was measured by ELISA.6. Statistical methodAll experiments were performed for five independent times. Data were expressed as the means±standard error and were analyzed for significant differences by unpaired Student’s t test and one-way ANOVA with SPSS 10.0. Differences were considered statistically significant if P value <0.05.Result1. The curve length, area, and density of A549 cells were changed after phagocytosisThe whole process of phagocytosis of apoptotic eosinophils by A549 cells was automatically recorded every one minute. The curve length, area and density of A549 cells before and after phagocytosis were measured by MetaMorph software. The results showed that the curve length and area of A549 cells were reduced after phagocytosis (P<0.001), while the density of A549 cells were increased (P<0.001).Phagocytosis was further confirmed by laser confocal scanning microscope.2. Dexamethasone promotes engulfment of apoptotic eosinophils by A549A549 epithelial cells recognized and ingested apoptotic eosinophils that were visualized as dark-staining peroxidase-positive cells. Effects of dexamethasone stimulation with various duration of preincubation on the engulfment of apoptotic eosinophils by A549 cells showed that the effect was not observed at shorter incubation time (< 1 h), but was evident at longer incubation time. The capacity of engulfment reached the highest at the 4h of preincubation (55%±2.2%) and reduced to the 38%±1.4% at the 24h of preincubation which was still significantly higher than that in control (0h).3. Dexamethasone treatment increased annexin I on cell surface of A549Western blot analysis showed the effect of dexamethasone treatment on the expression of annexin I on cell surface of A549. The result showed that the intensity was maximal at 4h after dexamethasone treatment and returned to the basal levels at 24h. Correlation analysis shown that the amount of cell surface annexin I was positively correlated with the capacity of engulfment induced by dexamethasone (r = 0.779, p<0.001).4. Increased phagocytosis of apoptotic eosinophils by A549 cells stimulated by treatment with dexamethasone was inhibited by annexin I mAbTo investigate the functional contributions of A549 cell surface annexin I, we tried to use the annexin I mAb to inhibit the phagocytosis of apoptotic eosinophils by A549 cells. The capacity of phagocytosis of A549 cells treated with dexamethasone and annexin I mAb (10μg/ml; 30 min at 37°C) was compared with that of A549 cells treated with dexamethasone only, previously shown to significantly enhance phagocytosis. The results showed that pretreatment with dexamethasone and annexin I mAb downregulated the phagocytosis of apoptotic eosinophils.5. Annexin I expressed on apoptotic eosinophil surface was involved in its phagocytosis by A549Apoptotic eosinophils were washed with EDTA to remove annexin I before presented to A549 cells. The result showed removal of annexin I with EDTA and preincubation with annexin I mAb inhibited phagocytosis (p<0.001).6. Dexamethasone-promoted uptake of apoptotic eosinophils did not change the proinflammatory factor secretion in A549 cellsThere was no difference in the release of proinflammatory factors and TGF-β1 between the A549 cells stimulated with apoptotic eosinophils and non-stimulated A549 cells. Conclusion1. The curve length, area, and density of A549 cells were changed after phagocytosis. The curve length and area of A549 cells were reduced after phagocytosis, while the densities of A549 cells were increased.2. Dexamethasone promotes engulfment of apoptotic eosinophils by A549 through annexin I.3. Dexamethasone-promoted uptake of apoptotic eosinophils did not change the proinflammatory factor secretion in A549 cells.更多还原