【作者】 屈飞；

【导师】 秦晓群；

【作者基本信息】 中南大学 ， 生理学， 2009， 博士

【摘要】 目的:在气道炎症或气道高反应（airway hyperresponsiveness,AHR）中,气道粘液分泌量异常增多,粘液组成成分也发生改变,容易形成气道粘液栓,是气道阻力增高的重要病理机制。气道上皮细胞粘液分泌的数量及组成成分取决于某些离子通道的功能活性及表达密度,其中囊性纤维化跨膜转导调节因子（Cystic fibrosis transmembraneconductance regulator,CFTR）尤为人们所关注。CFTR是一种磷酸化调控的上皮细胞Cl^-通道,主要位于气道上皮细胞顶侧膜,借助其对Cl^-的跨膜转运,在跨上皮盐类物质转运、水分流动和离子浓度调节中发挥重要作用。CFTR功能缺陷的患者分泌的粘液成分也与正常人很不一样,粘液包含有细菌感染产物、粘液脂质、肌动蛋白、蛋白酶等,使得其含有更多的非蒸发性的固体成分,增加粘液的稠度,降低粘液清除率和粘液运输效率,容易诱发气道炎症和形成气道粘液栓。基于以上的部分线索,本课题提出“气道上皮细胞CFTR门控功能及表达调节参与气道功能稳态,其缺陷可能与AHR形成机制有关”。方法与结果:1)氧化应激抑制支气管上皮细胞CFTR表达及信号机制我们用臭氧连续应激大鼠3天,每天1小时建立气道高反应动物模型,免疫荧光结果显示,正常大鼠支气管上皮顶膜及胞浆中可见较多的红色染色的CFTR,而臭氧应激的大鼠支气管上皮顶膜及胞浆中CFTR明显减少。我们应用臭氧攻击BECs 30min,建立氧化应激模型。Real-time PCR结果显示,臭氧应激4h后,CFTR mRNA表达明显下降。免疫荧光显示正常BECs胞膜有较强的CFTR染色,臭氧处理后,CFTR。在膜及胞浆均明显下降。Western blot结果显示臭氧应激4h后,CFTR蛋白明显下降。全细胞膜片钳结果发现,在没有激动剂的情况下,记录的CFTR cl^-电流很小,在加入5μM forskolin,记录出明显的CFTR cl^-电流,在forskolin的基础上,可观察到臭氧应激4h后的BECs CFTR cl^-电流下降。在随后的试验中,发现这种cl^-电流能被CFTR cl^-电流抑制剂glibenclamide阻滞,而不能被非CFTR cl^-电流抑制剂DIDS所阻断,说明记录出的cl^-电流是CFTR cl^-电流。为了研究氧化应激抑制BECs CFTR表达的信号机制,在臭氧应激前,BECs被JAK2激酶抑制剂AG490（10μmol/L）预处理4h,Real-timePCR结果发现臭氧应激后,AG490能增加CFTR mRNA的表达,AG490单独处理对CFTR mRNA的表达没有影响。免疫荧光和Western blot结果均显示AG490预处理能增加CFTR蛋白的表达。随后Western blot结果显示在未受臭氧处理的BECs,STAT1蛋白的磷酸化水平很小,在臭氧处理后,STAT1的磷酸化水平随时间依赖性增加。在前期实验中我们已发现臭氧应激后,小分子NO、CO、ROS增加。为了了解这些小分子是否参与臭氧诱导的STAT1的磷酸化。应用这些小分子抑制剂预处理BECs,免疫荧光和Western blot结果显示AG、ZnPP-9、NAC均降低了臭氧诱导的STAT1的磷酸化。我们还对信号分子抑制剂是否调节臭氧应激后CFTR的表达进行了研究。Real-time PCR和Western blot结果显示AG,NAC预处理能增加臭氧应激后CFTR的表达,而ZnPP-9则影响不明显。2) VIP激活支气管上皮细胞CFTR转运及通道活性CFTR转运涉及到四个过程,最终调节细胞和膜上CFTR的水平。1)CFTR合成及构想成熟后,从ER转运至胞膜表面;2)在细胞膜表面,CFTR通过小泡以及酪氨酸为基础的基序发生内吞作用,CFTR从胞膜内陷至内涵体;3)胞吞的CFTR CFTR循环回胞膜上;4)胞吐的CFTR发生降解。CFTR只有在胞膜上才能对Cl^-的跨膜转运,在跨上皮盐类物质转运、水分流动和离子浓度调节中发挥重要作用。为了解CFTR的转运过程,我们通过以下实验来观察。免疫荧光结果发现VIP作用以后,循环回胞膜的CFTR增多,VIP促进了CFTR向胞膜的循环。免疫共沉淀结果显示正常BECs既表达成熟型CFTR（BandC）,又表达非成熟型CFTR（Band B）。臭氧降低了成熟型CFTR的表达,而VIP则增加了成熟型CFTR的表达。Western blot结果显示臭氧降低了胞膜和总CFTR的蛋白水平,VIP增加了胞膜和总CFTR的蛋白水平,VIP促进了CFTR向膜的转运。随后我们研究VIP对BECs上CFTR依赖的cl^-转运的调控机制进行了研究。采用氯离子敏感荧光染料MQAE检测胞内cl^-浓度和氯化物的分泌,结果发现VIP具有与forskolin一样增加胞内cl^-浓度和氯化物的溢出的特性,并随浓度升高而增大。全细胞记录结果显示在VIP的作用下,CFTR Cl^-电流密度增加,并具有浓度依赖性,在10^-8mmol/L为最大半数有效剂量。CFTR Cl^-阻断剂glibenctamide能阻断该电流,而非CFTR Cl^-阻断剂不能阻断该电流。我们应用PKA阻断剂H89,结果发现H89部分阻断了VIP激活的CFTR Cl^-电流;应用PKC阻断剂H7（200μM）,同样结果发现H7部分阻断了VIP激活的CFTR Cl^-电流。在BECs上存在着多种VIP受体（VPAC）,我们应用(10^-8-10^-10M)VIP受体拮抗剂,结果发现VIP受体拮抗剂能剂量依赖性抑制VIP激活的CFTR Cl^-电流。3) CFTR基因表达调控研究为了进一步探讨臭氧应激后CFTR的基因表达调控机制,我们对臭氧应激条件下CFTR的转录因子调节谱进行了研究。实验首先用TESS软件对CFTR启动子区转录因子结合位点进行搜索,然后根据搜索结果,设计了6条探针,覆盖所有的转录因子结合位点,应用EMSA和ChIP的方法筛选了调控CFTR表达的转录因子。结果显示探针5、6有探针与蛋白结合形成的滞后带,能被100倍未标记探针所竞争,为特异性结合。通过突变探针结合实验和抗体超迁移实验,证实这2条探针结合的转录因子分别为Spl、ERa。臭氧应激4h后,降低了这两种转录因子的DNA结合活性。ChIP实验显示,Spl和ERa可特异性与CFTR启动子结合。紧接着,我们用基因定点突变技术观察了两种转录因子对CFTR启动子活性的影响,结果显示Spl与ERa的结合位点突变后,CFTR启动子活性下降,Spl与ERa结合位点同时突变,可几乎到达到臭氧应激对CFTR的抑制。随后,我们用反义寡核苷酸技术观察了两种转录因子对CFTR表达的影响,Western证实了两种ASOs的有效性,他们可分别阻断两种转录因子的蛋白表达。应用两种ASOs后,我们用real-time PCR和Western观察到,Spl和ERa ASO处理可降低CFTR的表达。通过进一步的研究,我们用EMSA观察到ERa与Spl的结合活性在0-1h之间激活,于应激后2h开始下降。real-time PCR检测CFTR的表达与臭氧应激后ERa和Spl的转录活性一致。我们还用免疫荧光观察到臭氧应激4h后,Spl核转位降低。臭氧应激4h后,Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录。综上所述:本研究观察到臭氧应激抑制了BECs上CFTR的表达及功能,这为本课题提出的非CF疾病的气道炎性疾病中CFTR的功能下降提供了依据。研究还观察到臭氧应激后,NO、ROS产生增多,参与并激活了STAT1的磷酸化,抑制了CFTR的表达,臭氧应激还使Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录。另外本研究研究了VIP激活了CFTR的转运及门控特性,PKA和PKC途径通过调节CFTR调节区上的磷酸化位点调节了CFTR的通道功能,为治疗包括CF疾病在内的气道炎性疾病因CFTR功能下调引起的黏液粘稠问题提供了选择。结论:臭氧应激抑制了BECs上CFTR的表达及功能。其机制如下:（1）臭氧应激抑制了CFTR的表达。其途径有早期信号分子NO和ROS的产生,进而激活JAK/STAT途径参与臭氧应激抑制CFTR的表达。臭氧应激还使转录因子Spl,ERa与CFTR的启动子区的结合活性及核转位降低,抑制CFTR转录;（2）臭氧应激抑制CFTR cl^-电流,VIP激活了CFTR cl^-电流,PKA和PKC途径参与了其调控过程;（3）VIP增加了CFTR向胞膜的转运。主要是通过增加CFTR循环回胞膜,增加175kD成熟型CFTR的表达及增加胞膜CFTR蛋白的表达这三个过程来实现的。更多还原

【Abstract】 Objectives:The change of mucosal fluid composition result in the decrease of mucosa cilia clearance rate,the increase abnormally of airway mucosal fluid secretory volume,a key pathophysiological process in airway hyperresponsive diseases such as asthma and bronchitis.Cystic fibrosis transmembrane conductance regulator（CFTR）,functions as a cAMP-regulated Cl^- channel which controls transepithelial electrolyte transport,fluid flow,ion concentrations in the airway.Loss of CFTR Cl^-function caused a lack of fluid secretion with excessive fluid absorption, which led to watery component reduction in airway surface liquid,mucous thickening,blockage of submucosal gland ducts,and impairment of mucociliary clearance that were followed with infection,inflammation,and ultimately,the tissue destruction characteristic of bronchiectasis.on account of above part cue,we propose that the gating and the expression regulation of CFTR in BECs participate in the homeostasis of airway function,which the defect of CFTR function could be related with AHR.Contents:1.Ozone stress down-regulates the expression of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial cellsWistar rat inhaled 1.5ppm ozone 1h per day for three days to establish a airway hyperresponsiveness animal model.BECs were cultured on slides in DMEM/F12 medium with or without 1.5ppm ozone for 30 minutes to establish oxitative stress model.To investigate abnormalities of cystic fibrosis transmembrane conductance regulator（CFTR） expression in chronic inflammatory airway diseases and its regulation mechanisms,the present study was designed to observe the expression of CFTR,CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR,immunofluorescence, western blot and whole cell patch-clamp.The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells （HBEC）.STAT1 signal pathway was checkedto investigate the signal mechanism.It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC,which could be influenced by some signaling molecules related to the early transduction of cellular stress.Furthermore,reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR.Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC.The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules,and subsequently transcription modulator STAT1.2.Activation of CFTR trafficking and gating by vasoactive intestinal peptide in Human bronchial epithelial cellsThe present study was designed to observe the trafficking of CFTR, and channel gating in Human bronchial epithelium cells（HBEC） by using confocal microscopy,western blot,immunoprecipitation and Whole-cell patch clamp.Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. During stimulation with VIP,apical extension of CFTR immunofluorescence into the cell was reduced significantly and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP raised cell surface and total CFTR.Compare with the augmented level of total CFTR,the surface CFTR increased well than that the total CFTR.Immunoprecipitation founded that CFTR band C had an increase markedly in HBEC treated with VIP,compared with the control group.We also observed an increase in the CFTR band B,the immature form of CFTR,which the extent was lower than that band C. VIP led to 4-fold increases in Cl-effiux in HBEC.Glibenclamide-sensitive and DIDS-insensitive CFTR Cl^- currents was consistently observed after stimulation with VIP(10^-8mol/L).The augmention of CFTR Cl^- currents enhanced by VIP(10^-8mol/L) was reversed,at least in part,by the protein kinase A（PKA） inhibitor,H-89 and the protein kinase C（PKC） inhibitor, H-7,suggesting PKA and PKC participated in the VIP-promoted CFTR Cl^-currents.3.Study gene expression modulation of CFTRTo probe the mechanisms of the induced expression of CFTR under ozone stress,six oligonucleotide probes corresponding to various regions of the CFTR promoter were used in EMSA studies.Two were found to have a decrease mobility shift with extracts from ozone-stressed cells. Based on the assay of antibody supershift,they were verified as Sp1 and ERα.By ChIP assay,Sp1 and ERαdecreaed the ozone-inducible DNA binding on the CFTR promoter.Next,site-directed mutagenesis technology and antisense oligonucleotide technology were used to observe the inhibitory effects of the two nuclear factors on CFTR promoter activation and expression.The results also showed that Sp1 and ERαdecreased the ozone-inducible DNA binding on the CFTR promoter and CFTR expression.The translocation of Sp1 was observed by immunofluorescence assay, which showed that Sp1 nuclear translocation was decreased after ozone exposure.The time courses of Sp1 and ERαactivation and inactivation, followed by CFTR expression were also examined.It was shown that ozone-depression CFTR expression and Sp1 and ERαbinding activity correlated during a-24 hour time course.In summary:In present study,we observed that ozone stress repressed the eapression and function of CFTR,which provided support for studies investigating CFTR function in inflammatory lung diseases other than cystic fibrosis.We also investigate that the production of NO,ROS were increased and participated in activation of STAT1 after ozone stress,which contributed to the inhibition of CFTR expression,the binding activity and the translocation were inhibited of Sp1,ERαafter ozone stress,which resulted in the repression of CFTR transcription.In addition,we revealed VIP activated the trafficking and gating,the CFTR channel was regulated by PKA and PKC pathway through the phosphorylation of CFTR regulatory domain,which offered a choose to cure inflammatory lung diseases.Conclusion:Ozone stress repressed the expression and function of CFTR in BECs. As follow were its mechanisms:（1） Ozone stress down-regulated the expression of CFTR.Its pathways had NO,ROS,JAK/STAT.Ozone stress depressed the binding activity and the translocation of Sp1,ERα,which resulted in the inhibition of CFTR transcription;（2） Ozone stress inhibitted CFTR cl^- current,VIP activated CFTR cl^-current.Its signal pathway had PKA and PKC;（3） VIP raised CFTR trafficking,which included enhance recycling of CFTR,augment the expression of mature CFTR and the protein expression of plasmalemma CFTR.更多还原