【作者】 刘志奇；

【导师】 刘立思；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉头颈外科， 2013， 博士

【摘要】 第一部分变应性鼻炎小鼠动物模型的建立目的：建立变应性鼻炎小鼠动物模型,探讨变应性鼻炎鼻腔粘膜随鼻内激发时间不同的组织学变化。方法：4～6周龄BALB/c小鼠,用卵清蛋白50μg、氢氧化铝凝胶5mg加生理盐水1ml配成混悬液进行腹腔注射基础致敏共7次；5%卵清蛋白生理盐水滴鼻进行鼻腔激发,按组分别连续激发10天,20天,30天。对照组用生理盐水腹腔注射和滴鼻。观察小鼠行为学变化,酶联免疫吸附法测试外周血OVA-sIgE的水平,HE染色观察鼻粘膜嗜酸性粒细胞浸润情况,检测结果采用SPSS16.0统计软件进行数据处理,组间比较用T检验。结果：造模结束后小鼠行为学评分分别为5.01±1.01、6.43+1.12、8.56+1.64,对照组0.85+0.23；鼻黏膜内EOS计数分别为45.91(45.91±5.80) pg/ml、48.81(48.81±3.49) pg/ml、51.38(51.38±3.19) pg/ml对照组0.69±0.12；血清OVA-sIgE浓度分别45.91(45.91±5.80)pg/ml.48.81(48.81±3.49)pg/ml51.38(51.38±3.19)pg/ml,对照组为41.71(41.71±2.58)pg/ml。T检验显示,实验组和对照组之间均存在显著性差异(P<0.05),AR模型组之间差异有统计学意义(P<0.05)。结论：变应性鼻炎小鼠动物模型造模成功,随着鼻内激发时间的延长,OVA-sIgE浓度和症状有升高趋势。第二部分TFH细胞在变应性鼻炎小鼠模型外周血中的流式细胞分析目的：流式细胞术分析TFH细胞在变应性鼻炎小鼠模型外周血的变化和意义。方法：变应性鼻炎小鼠模型成功建立后,用红细胞裂解液处理得到的外周血单个核细胞(PBMC)。CD4-Fitc和CXCR5-PE染色后用流式细胞仪分析检查外周血的CD4+CXCR5细胞的比例,ELISA分析血清总OVA-sIgE水平,用统计学软件SPSS16.0作T检验和Pearson相关性分析。结果：卵清蛋白激发AR模型组外周血中TFH细胞占外周血单个核细胞的百分比分别为7.94±2.66%、8.65±3.49%、9.77±3.24%,对照组为4.97±0.20%,经比较差异有显著性,前者明显高于后者(T-test,P<0.05)；AR模型组之间的百分比随着免疫激发时间延长的而增加,经比较差异有显著性(T-test, P<0.05)。Pearson相关性分析显示TFH细胞的表达水平与总OVA-sIgE水平呈正相关(r=0.87,P=0.021)。结论：TFH细胞在变应性鼻炎发病中有重要的作用,可能与IgE水平升高有关。第三部分IL-21在变应性鼻炎小鼠模型中的表达及与TFH之间的关系目的：探讨IL-21分子在变应性鼻炎小鼠模型中的表达及与OVA-sIgE、TFH细胞的关系。方法：变应性鼻炎BALB/c小鼠造模成功后,ELISA法测试外周血IL-21、OVA-sIgE的量,流式细胞术分析TFH细胞在外周血单个核细胞中的含量，SPSS16.0中Pearson相关性分析IL-21分子与各组数据之间的联系,均数间的比较用T检验。结果：IL-21在对照组为186.25±11.68pg/ml, AR模型1组为181.13±4.93pg/ml,AR模型2组为168.46±11.04pg/ml, AR模型3组为164.54±10.52pg/ml。各试验组和对照组小鼠外周血IL-21的表达经比较差异有显著性意义(T-test,P<0.05),试验组IL-21的表达水平要比正常组低,而且随着鼻内激发时间的延长,IL-21的表达水平逐渐下降；Pearson相关分析显示外周血IL-21的表达水平与CD4+CXCR5+T细胞比例呈负相关(r=-0.80,P=0.048),与血清总OVA-sIgE水平呈负相关关系(r=-0.86,P=0.031)。结论：IL-21与TFH细胞及OVA-sIgE的含量呈负相关,临床上可能对变应性鼻炎有一定的治疗作用。第四部分TFH相关因子在变应性鼻炎小鼠模型鼻黏膜中的表达目的：探讨TFH相关因子CXCR5及Bcl-6在变应性鼻炎小鼠模型鼻粘膜中的表达及可能意义。方法：小鼠变应性鼻炎模型造模成功后,用免疫组织化学方法分析CXCR5在小鼠鼻黏膜中的表达情况。并用Westernblot和PCR的方法对TFH关键转录因子Bcl-6进行分析,数据用医学统计学软件SPSS16.0处理及比较。结果：Western blot测试显示TFH关键转录因子的表达在实验组鼻黏膜明显升高,其相对密度值分别为0.67、0.84、0.98,而对照组鼻黏膜为0.53。鼻黏膜适时荧光定量PCR检测显示Bcl-6mRNA的表达也明显增高,其扩增倍数分别为1.63、1.77、1.85、1.92,而对照组鼻黏膜为0.90(T-test, P<0.05)。鼻黏膜CXCR5免疫组织化学分析的结果显示实验组鼻黏膜CXCR5+细胞数比对照组鼻黏膜中CXCR5+细胞数明显增多(T-test, P<0.05),广泛分布在粘膜及粘膜下层中。且随着鼻内激发时间的延长表达增多。结论：变应性鼻炎小鼠鼻粘膜中CXCR5及Bcl-6表达明显增强,可能参与了变应性鼻炎的发病过程。更多还原

【Abstract】 Part I Establishment of mouse model with allergic rhinitisObjective:To establish a mouse model with allergic rhinitis, explore tissue changes in nasal mucosa with challenge times.Methods:BALB/c mice,4to6weeks old with50μg ovalbumin, aluminium hydroxide gel5mg and lml saline sensitized by intraperitoneal injection of7times;5%ovalbumin solution dropped for nasal provocation, were continuous applied of10days,20days,30days. The control group was replaced by0.9%saline. To observe the mice behavior, the level of OVA-sIgE by ELISA, nasal mucosa infiltration of eosinophils by HE, the results was analyzed using SPSS16.0.Results:The mice behavior scores of experiment were5.01±1.01,6.43±1.12,8.56±1.64, control group0.85±0.23; nasal mucosal EOS counts were5.63±1.25,9.25±2.10,15.37±2.36,0.69±0.12in the control group; the serum OVA-sIgE concentration was45.91(45.91±5.80)pg/ml、48.81(48.81±3.49) pg/ml、51.38(51.38±3.19) pg/ml, in control group (41.71±2.58pg/ml). T test showed that there were significant differences between the experimental group and the control (P<0.05), and between the model groups (P<0.05).Conclusion:Allergic rhinitis mouse model was established successfully; more nasal challenges, the concentration of OVA-sIgE and the symptoms were more increased. Part II Flow cytometric analysis of TFH cells in allergic rhinitis mouse modelObjective:To investigate the expression and significance of TFH cells in allergic rhinitis mouse model.Methods:After allergic rhinitis mice model established successfully,peripheral blood mononuclear cells(PBMC) were obtained with red cell lysates which were analyzed using CD4-Fitc and CXCR5-PE by flow cytometry to get the proportion of CD4+CXCR.5+cells in peripheral blood, the total serum OVA-sIgE levels was tested by ELISA, the relationship was analyzed by Pearson correlation in SPSS16.0.Results:TFH cells in AR model groups were respectively7.94±2.66%、8.65±3.49%、9.77±3.24%, the control group was4.97±0.20%, the difference was significant analyzed by T-test(P<0.05);differences between the model groups showed to be significant (T-test, P<0.05) and Pearson correlation analysis showed that the expression level of TFH cells and total OVA sIgE levels were positive (r=0.87, P=0.021)Conclusion:TFH cells have an important role in the pathogenesis of allergic rhinitis, which might be associated with the elevated OVA-sIgE. Part III Relationships between IL-21and TFH in mouse model with allergic rhinitisObjective:To investigate the expression of IL-21and its relationships between OVA-sIgE and TFH cells in allergic rhinitis mouse model. Methods:Peripheral blood was collected from allergic rhinitis BALB/c mice to test IL-21and OVA-sIgE by ELISA quantity, the percentage of TFH cells in PBMC by flow cytometry. its relationships between them were analyzed by Pearson correlation in SPSS16.0.Results:The expressions of11-21were186.25±11.68pg/ml in the control,181.13±4.93pg/ml in group AR1,168.46±11.04pg/ml in group AR2,164.54±10.52pg/ml in group AR3. After statistical analysis, IL-21showed to be negative correction with CD4+CXCR5+T cell ratio((r=-0.80, P=0.048) and total serum OVA-sIgE levels (r=-0.86, P=0.031)Conclusion:IL-21was negative correction with CD4+CXCR5+T cell ratio and total serum OVA-sIgE levels; it might have some therapeutic effect on allergic rhinitis in clinic. Part IV Expression and significance of TFH related cytokines in mouse nasal mucosa with allergic rhinitisObjective:To investigate the expression and significance of TFH related factor CXCR5and Bcl-6in allergic rhinitis of mouse nasal mucosa.Methods:The expression of CXCR5in nasal mucosa was analysed by immunohistochemical method. The TFH transcription factor of Bcl-6was tested by means of Westernblot and PCR, data processing and compare were used by medical statistical software SPSS16.0.Results:Westernblot showed the expression of bcl-6factor was increased significantly in the experimental groups, the relative density values were0.67,0.84,0.98, while the control was0.53. PCR assay showed Bcl-6mRNA expression were significantly increased, the amplification were0.90,1.63,1.77,1.85,1.92individually (T-test, P<0.05). CXCR5+cells increased significantly from the control group to different experimental groups (T-test. P<0.05), and were widely distributed in the mucosa and submucosa.Conclussion:CXCR5+cells and Bcl-6expression increased in mice nasal mucosa,which might play a role in the pathogenesis of allergic rhinitis.更多还原