【作者】 王伟伟；

【导师】 郑鸣；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 [目的]1.原代大鼠鼻粘膜上皮细胞的纯化、培养及其鉴定。2.探讨环腺苷酸-蛋白激酶A (cyclic adenosine monophosphate-dependent protein kinase-A,cAMP-PKA)信号通路对大鼠鼻粘膜上皮细胞水通道蛋白5(aquaporin 5AQP5)调控机制。3.SD大鼠变应性鼻炎(Allergic rhinitis, AR)动物模型的建立及其评价。4.探讨核因子kappa B(Nuclear factor kappa B NF-κB)信号通路对AQP5表达的影响。5.探讨变应性鼻炎高分泌性的可能机制。6.探讨变应性鼻炎时NF-κB信号通路和cAMP-PKA信号通路之间的cross-talk。[方法]1.采用差速贴壁法和条件限定培养基,纯化和培养大鼠鼻粘膜上皮细胞。免疫细胞化学染色鉴定细胞类型并判断其纯度。2.PKA抑制剂H89(3μM)和cAMP激活剂forskolin (1μM)干预鼻粘膜上皮细胞12h和24h。CCK-8法检测H89和forskolin对上皮细胞生长的影响；免疫细胞化学染色、实时荧光定量PCR (Real-time PCR)及蛋白免疫印迹(western-blotting)检测各组鼻粘膜上皮细胞AQP5和p-CREB(Ser133)的表达。3.通过卵清蛋白(OVA)全身和局部致敏,建立大鼠变应性鼻炎模型,Wright’s染色检测模型组鼻腔分泌物和鼻粘膜组织嗜酸性粒细胞的表达；酶联免疫吸附测定法(ELISA)检测外周血IgE水平；行为学积分对动物模型进行评价。4. NF-κB抑制剂四氢化吡咯二硫代氨基甲酸酯(Pyrrolidinedithiocarbamate ammonium, PDTC 100mg/kg/d和50mg/kg/d)干预AR大鼠,免疫组织化学、western-blotting和Real-time PCR检测各组鼻粘膜组织NF-κBp65、p-CREB(Ser133)、AQP5、IL-1β、TNF-α及IL-6表达。5.H89和forskolin干预AR大鼠,免疫组织化学、Real-time PCR和western-blotting检测各组鼻粘膜组织,NF-κBp65. p-CREB(Ser133)、AQP5、IL-1β及TNF-α表达。6. Western-blotting检测各组鼻粘膜组织粘蛋白5AC(MUC5AC)和粘蛋白5B(MUC5B)表达。[结果]1.采用差速贴壁法和条件限定培养基,可成功纯化和培养鼻粘膜上皮细胞。免疫细胞化学染色鉴定鼻粘膜上皮细胞纯度可达95%。2.H89和forskolin干预对大鼠鼻粘膜上皮细胞生长无显著影响。H89(3μM)干预12h和24h后,p-CREB(Ser133)和AQP5阳性细胞数较对照组明显减少,p-CREB(Ser133)蛋白、AQP5蛋白及AQP5mRNA表达较对照组明显减少；Forskolin (1μM)干预12h和24h后,p-CREB(Ser133)和AQP5阳性细胞数较对照组明显增多,p-CREB(Ser133)蛋白和AQP5蛋白及AQP5mRNA表达较对照组明显增多。3.通过卵清蛋白全身和局部致敏建立SD大鼠AR模型,HE染色显示模型组鼻粘膜组织纤毛脱落、倒伏,血管、腺体增多,Wright’s染色显示模型组嗜酸性粒细胞浸润；外周血IgE水平增高；行为学积分较对照组明显增高。4.与对照组相比,模型组鼻粘膜组织NF-κBp65平均光密度值和蛋白表达明显增多,IL-1β、TNF-α及IL-6mRNA表达明显增多,p-CREB(Serl33)平均光密度和蛋白表达减少,AQP5平均光密度值、蛋白表达和mRNA表达减少；PDTC (100mg/kg/d and 50mg/kg/d)干预1d、3d和5d后,NF-KBp65平均光密度值和蛋白表达较模型组减少,IL-1β、TNF-α及IL-1βmRNA表达较模型组减少,p-CREB(Ser133)平均光密度值和蛋白表达较模型组增多,AQP5平均光密度值、蛋白表达和mRNA表达较模型组增多。5.H89 (5mg/kg/d)干预1d、3d和5d后,NF-κBp65平均光密度值和蛋白表达较模型组增多,IL-1β及TNF-αmRNA表达较模型组增多,p-CREB(Ser133)平均光密度和蛋白表达较模型组减少,AQP5平均光密度值、蛋白表达和mRNA表达较模型组减少；Forskolin (5mg/kg/d)干预1d、3d和5d后,NF-κBp65平均光密度值和蛋白表达较模型组减少,IL-1β及TNF-αmRNA表达较模型组减少,p-CREB(Ser133)平均光密度值和蛋白表达较模型组增多,AQP5平均光密度值、蛋白表达和mRNA表达较模型组增多。6.模型组MUC5AC和MUC5B蛋白表达较对照组明显增多；PDTC(100mg/kg/d and 50mg/kg/d)干预1d，3d和5d后,MUC5AC蛋白和MUC5B蛋白较模型组减少,H89(5mg/kg/d)干预1d,3d和5d后,MUC5AC蛋白和MUC5B蛋白表达较模型组增多。Forskolin(5mg/kg/d)干预1d,3d和5d后,MUC5AC蛋白和MUC5B蛋白表达较模型组减少。[结论]1.采用差速贴壁纯化法和条件限定培养基,可获得高纯度的鼻粘膜上皮细胞。2. cAMP-PKA信号通路通过CREB丝氨酸残基133位点磷酸化途径参与了鼻粘膜上皮细胞AQP5表达调控。3.通过全身和鼻腔局部OVA致敏,可成功建立SD大鼠变应性鼻炎动物模型。4. NF-κB信号通路可能通过抑制CREB(Ser133)磷酸化途径或者通过NF-κBp65与p-CREB (Ser133)竞争性结合CBP途径下调AQP5表达。5. NF-κB信号通路上调MUC5AC蛋白和MUC5B蛋白表达,可能引起变应性鼻炎高分泌性；AQP5的表达减少导致鼻腔分泌物清除或重吸收功能障碍,加速变应性鼻炎高分泌性。6. cAMP-PKA信号通路可抑制NF-κB信号通路。更多还原

【Abstract】 Objective1.The purification,culture and identification of rat primary nasal epithelial cells.2.To detect the mechanism by which Cyclic adenosine monophosphate-dependent protein kinase-A (cAMP-PKA) pathway regulates aquaporin 5(AQP5) on rat nasal epithelial cells.3.The establishment and evaluation of the rat model with allergic rhinitis (AR).4.To detect the effect of NF-κB pathway on the expression of AQP5 in the nasal mucosa of AR rats.5.To detect the possible mechanism on the hyper-secretion of AR.6. To detect the cross-talk between the NF-κB and cAMP-PKA pathways in AR.Methods1.The primary nasal epithelial cells were purified and cultivated by differential adhesion and defined medium,and were identificated by immunocytochemical staining,and estimated their purity2.The nasal epithelial cells were treated with PKA inhibitor H89 (3μM) or the cAMP activator forskolin (1μM) for 12h and 24h. CCK-8 was applied to detect the growth of the cultured nasal epithelial cells.AQP5 and p-CREB(ser133) were detected by immunocytochemical staining, western-blotting or Real-time PCR.3.The animal model of rats with allergic rhinitis was established by the general and local sensitization with ovalbumin (OVA).Eosinophils in the nasal secretions and the nasal mucosa of model group were detected by Wright’s staining.The level of IgE in peripheral blood was detected by enzyme-linked immunosorbent assay. Theanimal model with AR was evaluated by behavioral scores.4.AR rats were treated with NF-κB inhibitors pyrrolidinedithiocarbamate ammonium (PDTC 100mg/kg/d and 50mg/kg/d). the NF-κBp65,p-CREB(ser133),AQP5, IL-1β, TNF-αand IL-6 in the nasal mucosa were detected by immunohistochemistry, Real-time PCR or western-blotting.5.AR rats were treated with H89(5mg/kg/d) or forskolin(5mg/kg/d). p-CREB(ser133), NF-κBp65,AQP5,IL-1β,TNF-αand IL-6 in the nasal mucosa were detected by immunohistochemistry, Real-timePCR or western-blotting.6.The protein expression of MUC5AC和MUC5B of each group were detected by western-blotting.Results1.The rat nasal epithelial cells were purified and cultured by differential adherence and defined culture medium,which were identificated by immunocytochemical staining.The purity of the cells were more than 95%.2.H89 or forskolin made no difference on the growth of the rat nasal epithelial cells, After the treatment with 3μM H89,compared with group C,the positive cells of p-CREB(Ser133) and AQP5 decreased, the expression of p-CREB(Ser133) and AQP5 protein was decreased,and the mRNA level of AQP5 decreased;After the treatment with 1μM forskolin,compared with group C, the positive cells of p-CREB(Ser133) and AQP5 increased,and the expression of p-CREB(Ser133) and AQP5 protein was decreased,and the mRNA level of AQP5 increased.3.The model of SD rat with AR was established by by the general and local sensitization with ovalbumin (OVA).Compared with group C,the cilias on nasal mucosa tissue was off or flattened,the number of vessels,glands and eosinophile granulocytes increased,the level of IgE in peripheral blood was elevated,and the behavioral scores increased.4.Compared with group C,the mean optical density(MOD) and protein expression of NF-κBp65 of group M increased significantly,the mRNA level of IL-1β,TNF-αand IL-6 of group M increased,the MOD and protein expression of p-CREB of group M decreased,and the MOD, protein expression and mRNA level of AQP5 of group M decreased significantly.After the treatment with PDTC(100mg/kg/d and 50mg/kg/d) for ld,3d or 5d,compared with group M,the MOD and protein expression of NF-KBp65 decreased significantly,the mRNA level of IL-1β,TNF-αand IL-6 decreased,the MOD and protein expression of p-CREB(ser133) increased,and the MOD,protein expression and the mRNA level of AQP5 increased significantly.5.After the treatment with H89(5mg/kg/d) for 1d,3d or 5d,compared with group M,the MOD and protein expression of NF-κBp65 increased significantly,the mRNA level of IL-1β,TNF-αand IL-6 were up-regulated,the MOD and protein expression of p-CREB(Ser133) were reduced, the MOD,and protein expression and mRNA level of AQP5 decreased.After treatment with forskolin (5mg/kg/d) for for 1d,3d or 5d,compared with group M,the MOD and protein expression of NF-κBp65 decreased significantly,the mRNA level of IL-1β,TNF-αand IL-6 were down-regulated,the MOD and protein expression of p-CREB were elevated,the MOD,protein expression and mRNA level of AQP5 increased.6.The protein expression of MUC5AC or MUC5B in group M increased significantly compared with group C,but decreased after the treatment with PDTC(100mg/kg/d and 50mg/kg/d) for 1d,3d or 5d.After the treatment with H89 (5mg/kg/d)for 1d,3d or 5d, The protein expression of MUC5AC and MUC5B protein expression increased significantly,while they decreased in a time-dependent manner after the treatment with forskolin(5mg/kg/d) for ld,3d or 5d.Conclusions1.Rat nasal epithelial cells with high purity of more 95% were cultured by differential adherence and defined culture medium.2.The cAMP-PKA pathway is involved in the regulation of AQP5 in rat nasal epithelial cells by phosphorylating CREB at serine 133.3.The model with allergic rhinitis can be successfully established by general and local sensitization with OVA.4.The NF-κB pathway can down-regulate the expression of AQP5 by inhibiting CREB phosphorylation at serine 133 or by competitive binding to CBP with p-CREB(Serl33).5.The NF-κB pathway can up-regulate the protein expression of the MUC5AC and MUC5B,which maybe be involved in the hyper-secretion of AR, and the decrease of AQP5 results in the clearance dysfunction of the secretions,which promotes the hyper-secretion of AR 6.The cAMP-PKA pathway can inhibit the NF-κB pathway.更多还原