【作者】 王炯；

【导师】 刘荣玉；

【作者基本信息】 安徽医科大学 ， 老年医学， 2008， 博士

【摘要】 研究背景中性粒细胞及巨噬细胞是气道炎症性疾病的主要效应细胞。中性粒细胞通过吞噬、释放多种生物活性物质等发挥气道防御功能。在完成其使命后中性粒细胞将走向凋亡。巨噬细胞有效地吞噬清除凋亡的中性粒细胞对急性炎症的消退、恢复机体的稳态是至关重要的。但目前对巨噬细胞吞噬凋亡中性粒细胞的动态过程以及巨噬细胞吞噬凋亡中性粒细胞后的命运转归还知之甚少。近年研究表明:凋亡细胞清除障碍与气道慢性炎症性疾病进行性恶化、系统性红斑狼疮以及其他一些自身免疫性疾病有关。增强巨噬细胞吞噬清除凋亡的细胞将是这些疾病治疗的新策略。CpG-寡核苷酸是人工合成的含未甲基化的CpG二核苷酸基序的寡核苷酸序列,和细菌DNA一样具有强烈的免疫刺激作用,能激活哺乳动物巨噬细胞、树突状细胞等多种免疫细胞。但激活的巨噬细胞吞噬凋亡的中性粒细胞的功能是否增强以及其机制如何目前尚不清楚。目的实时动态观察巨噬细胞吞噬凋亡中性粒细胞的全过程;明确吞噬凋亡中性粒细胞后巨噬细胞的命运转归;探寻CpG-寡核苷酸和细菌-DNA是否具有增强巨噬细胞吞噬凋亡中性粒细胞中的功能及其可能的机制。方法1.中性粒细胞的分离及凋亡诱导10ml肝素抗凝的正常人外周血,红细胞裂解液去除红细胞,进一步采用Percoll不连续密度梯度离心法分离中性粒细胞,培养20h后中性粒细胞自发衰老而凋亡,流式细胞术进行凋亡评估;2.巨噬细胞吞噬凋亡中性粒细胞及吞噬诱导激活巨噬细胞死亡的实时动态观察巨噬细胞接种于35mm~2培养皿中生长过夜。然后置于具有加热系统的倒置显微镜载物台上,加入凋亡中性粒细胞后采用生物显微电影术实时动态观察记录巨噬细胞吞噬凋亡中性粒细胞的全过程并追踪观察吞噬后巨噬细胞继发性形态学改变;3.透射电镜分析吞噬后巨噬细胞的死亡方式巨噬细胞与凋亡中性粒细胞相互作用60分钟后洗去未吞噬的中性粒细胞,固定收集巨噬细胞,制备电镜标本,透射电镜观察分析;4.激光共聚焦扫描显微镜分析吞噬后巨噬细胞的死亡巨噬细胞与凋亡中性粒细胞相互作用60分钟后洗去未吞噬的中性粒细胞,分别采用MDC/AO、AO及AO/EB染色,激光共聚焦扫描显微镜下分析观察巨噬细胞死亡方式并进行各种不同死亡方式计数定量分析;5.吞噬凋亡中性粒细胞后巨噬细胞培养上清液IL-6和TNF-a测定巨噬细胞与凋亡中性粒细胞相互作用60分钟后洗去未吞噬的中性粒细胞,继续培养24小时,在规定的时间点收集上清,采用放免法测定IL-6和TNF-a浓度;6.CpG-DNA处理前后巨噬细胞吞噬凋亡中性粒细胞的定量分析接种RAW264.7细胞于24孔培养板中生长过夜并用CpG-DNA处理24小时。吞噬试验前,把长满RAW264.7细胞的玻片转移到用Parafilm膜包被好的12孔板中。把凋亡的中性粒细胞悬浮到DMEM中,加入到玻片上37℃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中性粒细胞后,接着用4%的多聚甲醛固定,o-dianisidine进行染色,进行计数分析;7.MTT分析巨噬细胞活力接种巨噬细胞于96孔板培养板中生长过夜,CpG-DNA处理24小时后用PBS洗2次,加入180μl的新鲜培养基和20μl的MTT(5 mg/ml)继续培养4小时;吸去上清后加150μl DMSO,摇床缓慢振荡10分钟完全溶解MTT结晶,利用酶标仪测定样本在波长570nm处的光密度OD值;8.总TLR9表达的Western Blot分析2×10~6巨噬细胞接种于35mm2培养皿中生长过夜,采用CpG-DNA处理24小时后裂解细胞提取总蛋白,接着进行Western Blot分析;9.流式细胞术分析巨噬细胞膜表面TLR9表达2×10~5巨噬细胞接种于24孔培养板中生长过夜,CpG-DNA处理24小时后收集巨噬细胞,与适当浓度的TLR9抗体及相应的二抗孵育后流式细胞仪测定细胞表面相对荧光强度。10.统计学方法所有试验分3～5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.我们首次动态观察了巨噬细胞吞噬凋亡中性粒细胞的全过程,发现巨噬细胞吞噬凋亡中性粒细胞过程也可分为识别粘附、内吞、消化和残体外排等阶段。同时观察记录了吞噬凋亡中性粒细胞后部分巨噬细胞被诱导激活死亡的动态过程,2.透射电镜、激光共聚焦扫描显微镜显示吞噬后的巨噬细胞死亡方式为自噬、凋亡和胀亡等,其中自噬率为8.00±2.00%、凋亡率为12.33±2.08%、胀亡率为3.66±1.50%,主要死亡方式为自噬和凋亡。3.吞噬凋亡中性粒细胞后巨噬细胞培养上清液中细胞因子IL-6、TNF-a浓度升高。4.同时我们发现细菌DNA和CpG-寡核苷酸具有增强巨噬细胞吞噬凋亡中性粒细胞的功能,这种增强功能可以被CpG-DNA拮抗剂—氯喹抑制;5.这种增强功能与巨噬细胞TLR9蛋白表达增高相一致;6.氯喹并不能抑制CpG-DNA刺激引起的TLR9的表达。结论:1.巨噬细胞吞噬凋亡中性粒细胞的过程可以分为识别粘附、内吞、消化、残体外排等不同的阶段;吞噬凋亡中性粒细胞也可以诱导激活巨噬细胞死亡;2.吞噬凋亡中性粒细胞后巨噬细胞被诱导激活有凋亡、自噬、胀亡等不同的死亡方式,吞噬凋亡中性粒细胞后的巨噬细胞死亡在免疫应答及炎症性疾病中有重要作用。3.细菌DNA和CpG-寡核苷酸可以通过TLR9增强巨噬细胞吞噬凋亡中性粒细胞的功能;增强巨噬细胞吞噬凋亡中性粒细胞功能同样需要内体的成熟/酸化。更多还原

【Abstract】 BackgroundsMacrophages and neutrophils are now recognized as the major effector cells in the airway inflammatory diseases.By infiltrating into the bronchial mucosa,the neutrophils can effectively bind and kill microorganisms that may cause damages to tissue,and then they timely undergo a vigilantly controlled programmed cell death known as apoptosis.Clearance of these apoptotic neutrophils by macrophages is important for the successful resolution of acute inflammation and tissue homeostasis. However,the process of phagocytosis of apoptotic neutrophils by macrophages and the fates of macrophages after their ingestion have not been unveiled.There are growing evidences that defective handling of apoptotic cells by macrophages plays a pivotal role in the development of chronic airway inflammatory diseases,systemic lupus erythematosus(SLE) and other autoimmune diseases. Strategies to improve clearance of apoptotic neutrophils may have therapeutic significance.Bacterial genomic DNA and synthetic oligonucleotide containing unmethylated cytosine followed by guanine(CpG-ODN) have been shown to stimulate immunocompetent cells,including macrophages/monocytes,dendritic cells (DCs),and B cells.Whether they can enhance the ability of macrophages to engulf apoptotic neutrophils and the potential mechanisms are still unclear.ObjectivesTo measure the dynamic process of phagocytosis of apoptotic neutrophils by macrophages in real time;to identify the fates of macrophages after their ingestion of apoptotic neutrophils;to investigate the role of CpG-ODN and bacterial DNA in the process of ingestion of apoptotic neutrophils by macrophages and the potential mechanisms.Methods1.Neutrophil isolation and apoptosis inductionAfter removal of red blood cells using red blood cell lysis buffer,the neutrophils were isolated by the Percoll discontinuous density gradient centrifugation.Apoptosis was assessed using flow cytometry after culturing for 20 hours2.Live cell imaging of Raw264.7 cells engulfing apoptotic neutrophils and phagocytosis induced cell death in macrophageRAW264.7 Cells grown in 35 mm~2 dishes were put into a heated-plate system on the stage of an inverted microscope equipped with a CCD camera controlled by SPOT RT software.Then apoptotic neutrophils were put into the dishes and allowed them to interact with each other.The dynamic process of phagocytosis of human apoptotic neutrophils by RAW264.7 cells in real time was recorded automatically every 30 seconds for a period of 5—12h.3.Form of macrophage cell death analyzed by transmission electric microscope After interacting with apoptotic neutrophils for 60 minutes and washing away the non-ingested ones,the macrophages were fixed,scraped and pelleted by centrifugation.Ultrathin sections were stained with uranyl acetate and lead citrate and were observed under a transmission electron microscope4.Form of macrophage cell death analyzed by confocal microscopeAfter interacting with apoptotic neutrophils for 60 minutes and washing away the non-ingested ones,the macrophages were stained with MDC/AO,AO,AO/EB respectively for 15min.Then the cells were scanned using confocal microscopy within 1 hour. 5.Quantification of IL-6 and TNF-a productionAfter interacting with apoptotic neutrophils for 60 minutes and washing away the non-ingested ones,the macrophages were cultured for additional 24h.The supernatants were collected at the indicated times points and IL-6 and TNF-a were assayed by radio-immuno assay(RIA).6.Quantification of uptake of apoptotic neutrophils by RAW264.7 cells treated with CpG-DNARAW264.7 cells were grown in 24-well plates and treated with CpG-ODN in the presence or absences of chloroquine for 24h.The cover-slips with RAW264.7 cells were transferred to 12-well plates coated with parafilm membrane.Apoptotic neutrophills were added to the coverslip with RAW264.7 cells for 60 minutes.After washing away the noningested neutrophils,the ingested neutrophils were visualized by o-dianisidine staining.RAW264.7 cells were counter-stained with haematoxylin. The cells were assayed and photographed.At least 200 RAW264.7 cells were counted in randomly selected fields.The proportion that had ingested one or more neutrophils was expressed as a percentage.7.Cell viability assay by MTTRAW264.7 cells were plated in 96 wells and incubated overnight.After 24 hours stimulation of CpG-ODN and B-DNA in the presence or absences of chloroquine for 2h ahead,Cells were then washed twice in PBS,and 180μl of fresh medium and 20μl of MTT(5 mg/ml) were added to each well,followed by incubation for an additional 4 h.The supematants were removed,and 150μl of DMSO was added to each well.MTT crystals were completely solubilized with libration for 10 min and optical density(OD) was measured at wavelengths A570 nm.The percentage of cell viability was calculated.8.Western blot analysis of total TLR9Macrophages(2×10~6) were treated with CpG-ODN and B-DNA for 24h in the presence or absence of chloroquine for 2h ahead.Then the cells were harvested and lysed with lysis buffer.Cell lysates were then subjected to Western blotting for TLR9 9.Flow cytometry analysis of surface TLR9For measuring surface expression of TLR9,cells were analyzed with a flow cytometry after 24 hours treatment with CpG-ODN and B-DNA in the presence or absence of 2μg/ml chloroquine for 2h ahead.The cells were incubated with the appropriately diluted anti-TLR9 mAb,followed by incubation with fluorescein isothiocyanate-conjugated secondary mAb.Then macrophages were analyzed for TLR9 on the flow cytometer10.Statistical analysisAll experiments were performed for three or 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.Results1.We measured for the first time the dynamic process of phagocytosis of apoptotic human neutrophils by RAW264.7 cells in vitro.We staged four steps of phagocytosis as the recognition and tethering,internalization,ingestion and exocytosis steps. Furthermore,we found that after phagocytosis,some macrophages might undergo a characteristic sequence of morphological changes,beginning with cell shrinkage, followed by membrane blebbing,and ultimately concluding in cell membrane rupture.2.The dead macrophages consisted of autophagy,apoptosis and oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes.The percentages of autophagic,apoptotic,oncotic macrophages were 8.00±2.00%,12.33±2.08%,and 3.66±1.50%respectively.3.The concentration of IL-6 and TNF-a in the supernatants of cultured macrophages was increased after interacting with apoptotic neutrophils.4.We found that the ability of macrophages to ingest apoptotic neutrophils was up-regulated by CpG-ODN and B-DNA treatment but the effect was abolished by chloroquine pre-treatment. 5.Increased uptake of apoptotic neutrophils was consistent with up-regulation of total and surface Toll-like receptor-9 expression in macrophages.6.Chloroquine couldn’t down-regulate the total and surface TLR9 expression in macrophages up-regulated by B-DNA and CpG-ODN treatment.Conclusions1.The phagocytosis of apoptotic neutrophils can also be divided into the recognition and tethering,internalization,digestion and exocytosis stages and the phagocytosis of apoptotic neutrophils can induce cell death in macrophages.2.After ingestion of apoptotic neutrophils,macrophages may undergo autophagy, apoptosis,oncosis that may play an important role in the pathogenesis and progression of inflammatory diseases.Autophagy of macrophage after ingestion of apoptotic cell may be one of the new mechanisms in immune response and inflammatory diseases.3.CpG-ODN and B-DNA can act through TLR9 to enhance the macrophages uptake of apoptotic neutrophils;enhancement of uptake of apoptotic neutrophils also requires endosomal maturation/acidification.更多还原