【作者】 张敏；

【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， 2012， 博士

【摘要】 目的：1．建立大鼠变应性鼻炎（Allergic rhinitis，AR）动物模型2．检测TLR2和TLR4亚型在正常及模型大鼠鼻粘膜上的表达情况3．探讨AR发病中TLR-NF-κB信号通路的可能机制4．探讨不同浓度Toll样受体4激动剂LPS诱导NF-κBp50高表达对AR发病的影响5．培养鼻粘膜上皮细胞并检测TLR4、NF-κBp50的表达6．应用培养的鼻粘膜上皮细胞，探讨LPS诱导NF-κBp50高表达的作用7．初步探讨雷公藤多甙干预变应性疾病的可能机制方法：1．通过卵清蛋白OVA(0.3mg/1mL/只)全身腹腔注射及局部喷鼻致敏，建立大鼠变应性鼻炎动物模型，HE染色鼻粘膜组织形态学改变及鼻腔、鼻粘膜组织嗜酸性粒细胞、中性粒细胞数计数含量，ELISA酶联免疫吸附测定法检测组织IgE水平，并行行为学评估动物模型。2.应用脂多糖LPS及肽聚糖PGN局部喷鼻干预，免疫组织化学染色法和Real-Time PCR检测各组鼻粘膜组织TLR2和TLR4的表达。3.施加脂多糖LPS局部喷鼻干预，免疫组织化学染色法和Real-Time PCR检测各组鼻粘膜组织细胞因子IL-4、IFN-γ、TLR及NF-κB p50的表达。4.应用不同浓度脂多糖LPS局部喷鼻干预，免疫组织化学染色法和或Real-Time PCR、Western-Blot检测各组鼻粘膜组织IL-4, IFN-γ, TLR，IgE及NF-κB p50的表达。5.采用差速贴壁法并施加条件限定培养基，分离纯化并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。6.应用培养的大鼠鼻粘膜上皮细胞，施加卵清蛋白OVA及脂多糖LPS干预，免疫组织化学染色法和Real-Time PCR检测各组鼻粘膜培养细胞的TLR及NF-κB p50的表达。7.通过施加雷公藤多甙干预，检测雷公藤多甙对TLR、NF-κB的表达及细胞因子Th1/Th2平衡的影响。实验的数据应用SPSS13.0统计软件包进行统计学处理，计量资料以x±s表示，组间比较采用单因素方差分析，P<0.05为差异有统计学意义。结果：1．通过卵清蛋白OVA(0.3mg/1mL/只)全身腹腔注射及局部喷鼻致敏，模型组Wright’s染色显示模型组大鼠鼻粘膜组织粘膜增厚显著，小血管扩张明显，组织中大量嗜酸性粒细胞浸润，ELISA酶联免疫吸附测定法检测组织Th2细胞因子IL-4及IgE水平较正常组明显升高，变应性鼻炎行为学积分显著高于正常组。2．应用脂多糖LPS及卵清蛋白OVA局部喷鼻干预，免疫组织化学染色法和Real-Time PCR显示各组鼻粘膜组织均有TLR及NF-κB p50的表达，且TLR及NF-κB p50的表达具有相关性。3.应用脂多糖LPS及肽聚糖PGN局部喷鼻干预，免疫组织化学染色法和Real-Time PCR检测显示各组鼻粘膜组织TLR2和TLR4的表达相对增强，中性粒细胞含量增多，鼻粘膜炎症反应明显。4.应用不同浓度脂多糖LPS局部喷鼻干预，免疫组织化学染色法和Real-Time PCR检测显示各组鼻粘膜组织IL-4, IFN-γ, TLR，IgE及NF-κB p50的表达与LPS浓度具有相关性。NF-κB抑制剂PDTC组NF-κB p50的表达相对抑制，Th细胞因子的表达相对减少。5.采用差速贴壁法分离纯化大鼠鼻粘膜上皮细胞，施加条件限定培养基纯化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及其纯度可达90％以上。6.应用培养的大鼠鼻粘膜上皮细胞，施加卵清蛋白OVA及不同浓度脂多糖LPS干预，免疫组织化学染色法和Real-Time PCR检测各组鼻粘膜培养细胞均有TLR及NF-κB p50的表达，且均有浓度相关效应。PDTC组NF-κB p50的表达相对减少，Th1/Th2细胞因子的比例较模型组增大，IgE的表达相对减少。7.雷公藤多甙降低TLR及NF-κB p50的表达，IL-4, IFN-γ表达均减少，Th1/Th2细胞因子的比值较模型组明显减小，IgE的表达相对减少。结论：1.通过OVA全身多次基础致敏、局部喷鼻激发能够成功地建立大鼠变应性鼻炎的动物模型。2．大鼠鼻粘膜组织TLR有TLR2和TLR4亚型的分布，正常鼻粘膜有较高的TLR2亚型的分布；TLR4亚型在AR发病中变化明显，对变应性鼻炎的发病发挥重要诱导作用。3．TLR在AR的发病发病中发挥诱导作用，变应原可能通过TLR-NF-κB信号通路诱导变应性疾病的发生发展。4． Toll样受体4激动剂LPS可诱导NF-κBp50的高表达，具浓度相关效应；高浓度LPS可诱导NF-κBp50的高表达，影响Th1/Th2平衡，相对抑制IgE的表达，产生免疫偏移作用，对AR发病有一定的影响。5．采用差速贴壁法可分离纯化大鼠鼻粘膜上皮细胞，施加条件限定培养基可纯化培养大鼠鼻粘膜上皮细胞。6．在成功培养的鼻黏膜上皮细胞，高浓度Toll样受体4激动剂LPS可诱导NF-κBp50的高表达，影响Th1/Th2平衡，产生免疫偏移作用，对AR发病有一定的影响。7.雷公藤多甙降低TLR的表达，调节TLR及NF-κB p50的表达，纠正Th1/Th2细胞因子的比例，降低IgE的表达，影响变应性鼻炎的发生发展。更多还原

【Abstract】 Objective:1. To establish the model of allergic rhinitis (AR) in rats.2. To assay the expression of TLR2and TLR4subtype in nasal mucosa for ordinaryand model rats.3. To discuss probable mechanism of TLR-NF-κBp50pathway cascaded in AR.4. To study the effect of TLR4agonist lipopolysaccharide (LPS) induced highexpression NF-κBp50with different concentration in AR.5. To grow primary nasal mucosa epithelial cells for detection of high expression ofTLR4, NF-κBp50.6. To study the effect of TLR4agonist lipopolysaccharide (LPS) induced highexpression NF-κBp50in primary culture nasal mucosa epithelial cells.7. To discuss probable mechanism of Tripterygium glycosides-interference in AR.Methods:1. Model of allergic rhinitis (AR) in rats was established by intraperitoneal injectionand nasal topic delivery of ovalbumin (OVA) in concentration of0.3mg/1mL/rat.By HE staining method, the change in histo-morphology of nasal musosa wasobserved and the number of neutrophil and eosinophile granulocyte in nasalcavity and mucosa was counted. The tissue IgE level was determined by ELISAand praxiological change on AR rat mode was assessed.2. After the model was interfered by nasal delivery of LPS and PNG, the expressionof TLR2and TLR4in nasal mucosa was detected by immunohistochemistry andReal-Time PCR.3. After the model was interfered by nasal delivery of LPS, the expression ofcytokines IL-4, IFN-γ, TLR and NF-κB p50in nasal mucosa was detected by immunohistochemistry and Real-Time PCR.4. After the model was interfered by nasal delivery of LPS in different concentration,the expression of cytokines IL-4, IFN-γ,TLR and NF-κB p50in nasal mucosawas detected by immunohistochemistry, Real-Time PCR and/or Western-Blot.5. Nasal mucosa epithelium cells of rats were cultivated by separation andpurification using selective plating technique and conditional medium.6. Based on cultured nasal mucosa epithelium cells of rats and the interference ofsaid cells by OVA and LPS, the expression of TLR and NF-κB p50in nasalmucosa was detected by immunohistochemistry and Real-Time PCR.7. Based on Tripterygium glycosides-interference of cultured nasal mucosaepithelium cells of rats, the expression of TLR and NF-κB p50, and equilibriumbetween cytokines Th1and Th2was detected.All data were represented as means±SEM. Statistical analysis was performed usingone-way ANOVA and Repeated measures with SPSS13.0software. A probabilityvalue of less than0.05was considered significant.Results:1. After intraperitoneal injection and nasal topic delivery of ovalbumin (OVA) inconcentration of0.3mg/1mL/rat, based on Wright’s staining of various modelgroups, a dramatic thickening in nasal mucosa of rats, an obvious hemangiectasisand an infiltration of EOS in large amount was observed. A significant increase ofcytokines TH2, IL4and IgE levels in tissue was detected by ELISA. Thepraxiological score of group AR was much higher than that of ordinary group.2. After the model was interfered by nasal delivery of OVA and LPS, the expressionof TLR2and NF-κB p50in nasal mucosa was detected in each group byinmmunohisocheminstry and Real-time PCR, furthermore the relationshipbetween TLR2and NF-κB was presented.3. After the model was interfered by nasal delivery of LPS and PGN, the expressionof TLR2and TLR4in nasal mucosa was relatively up-regulated byinmmunohisocheminstry and Real-time PCR. Increasing amount of Neurphile andintensity of inflammination in nasal mucosa were found.. 4. After the model was interfered by nasal delivery of LPS, the expression of IL-4, IFN-γ, TLR,IgE and NF-κB p50were correlated with the concentration of the LPS. In the group ofPDTC (an inhibitor of NF-κB), decreased expression level of NF-κB p50, decreasedexpression of Th cytocines and decreased expression level of IgE was found.5. After being cultivated by separation and purification using selective platingtechnique and conditional medium, the nasal mucosa epithelium cells identified byIHC Staining is more than90%.6. Based on cultured nasal mucosa epithelium cells of rats and the interference ofsaid cells by OVA and different concentration of LPS, the expression of TLR andNF-κB p50in nasal mucosa was presented by the method ofimmunohistochemistry and Real-Time PCR. In the group of PDTC, In the groupof PDTC, relatively decreased expression level of NF-κB p50, increased ratio ofTh1/Th2and relatively decreased expression level of IgE was found.7. Based on Tripterygium glycosides-interference of cultured nasal mucosaepithelium cells of rats, the expression of TLR，NF-κB p50, IL-4, IFN-γ, and IgEwere down-regulated. The ratio between cytokines Th1and Th2was higher thanthe modle group.Conclusion:1. Model of AR in rats could be successfully established by intraperitoneal injectionand nasal topic delivery induction with OVA.2. TLR in nasal mucosa of ordinary rats was existed in two subtypes: TLR2andTLR4. The TLR2was widely distributed in normal rat nasal tissue. The TLR4wasdramatically changed in AR model rat, which may cast a principal role ininitiating AR.3. TLR may initiating the AR disease. Allergen may initiating the progression of ARvia TLR-NF-κBp cascade.4. The TLR4agonist LPS could give rise to the up-regulation of NF-κBp50with aconcentration correlated effect. High concentration of LPS could increased theexpression level of NF-κBp50, alter the equilibrium between cytokines Th1andTh2. 5. The nasal mucosa epithelium could be obtain by using selective plating technique,and purified by adding conditional medium.6. In successfully cultivated nasal epithelium, the high concentration TLR4agonistLPS may up-regulate the expression level of NF-κBp50, then interfering theequilibrium cytokines Th1and Th2,.7. Tripterygium glycosides could alter the progression of AR by regulating the TLRexpression, NF-κBp50and the ratio of Th1/Th2.更多还原