【作者】 李化兵；

【导师】 韩秀珍； 王金荣；

【作者基本信息】 山东大学 ， 儿科学， 2008， 硕士

【摘要】 目的（1）探讨条件免疫反应（CIR）对小鼠支气管哮喘的作用机制（2）探讨条件免疫反应（CIR）对小鼠支气管哮喘的疗效方法选4-6周龄的雌性BALB/C小鼠,用卵清蛋白（OVA）腹膜下注射致敏,OVA雾化吸入激发,建立支气管哮喘模型,健康对照组用PBS进行相同操作。将模型小鼠随机分为7组,每组15只:A组:CIR₁组,以声音（85分贝）为条件刺激,普米克令舒加万托林雾化吸入为非条件刺激,两者结合7次（7天）后可建立条件免疫反应,此后每日再现条件刺激,每周条件刺激再与非条件刺激结合一次,共20周;B组:CIR₂组,以糖精水为条件刺激,其余操作同CIR₁组;C组:常规治疗组,给予普米克令舒和万托林雾化吸入治疗,1次/日,共20周;D组:常规治疗减量组,给予普米克令舒和万托林雾化吸入,1次/日,共7天,之后1次/周,共20周;E组:单纯声音刺激组,单纯给声音刺激20周;F组:单纯糖精水刺激组,单纯给糖精水刺激20周;G组:空白对照组,PBS雾化吸入20周。以上7组每天给予哮喘激发一次,其中各治疗组（A、B、C、D）在治疗间隔1小时以后给予激发。健康对照组为H组,用PBS激发20周。治疗20周后,分离小鼠脾脏单个核细胞,加入CD4⁺磁珠,利用分选器、分离柱分选出CD4⁺T淋巴细胞,经PMA、离子霉素和莫能霉素刺激培养6小时后,用流式细胞技术检测CD4⁺T细胞胞膜上烟碱型乙酰胆碱受体alpha7（nAChRα7）与胞浆内IL-4、IFN-γ及IL-17表达量,分析nAChRα7与IL-4、IFN-γ及IL-17的相关性。结果（1）治疗20周后,哮喘组（E、F、G组）及减量治疗组（D组）比健康对照组（H组）及各治疗组（A、B、C组）体重明显降低,症状加重（P＜0.01）。（2）治疗组（A、B、C组）比空白对照组（G组）肺泡灌洗液中嗜酸性粒细胞占白细胞的百分数明显减少。A、B、C组分别是:8.67±1.23%,9.13±0.99%,8.40±1.12%,G组是16.2±1.32%,t₁=16.13,t₂=16.58,t₃=17.44,P＜0.01;（3）治疗组（A、B、C组）与空白对照（G组）组相比较,CD4⁺T细胞表面nAChRα7表达降低。A、B、C组分别是:12.30±0.96%,12.6±1.14%,14.72±0.94%,G组是15.35±1.67%,t₁=7.81,t₂=6.51,t₃=7.62,P＜0.01;（4）治疗组（A、B、C组）与空白对照（G组）组相比较,IL-4表达降低。A、B、C组分别是:5.25±0.97%,4.73±0.80%,5.31±1.18%,G组是12.26±1.31%,t₁=16.68,t₂=19.03,t₃=15.30,P＜0.01;（5）治疗组（A、B、C组）与空白对照（G组）组相比较,IL-17表达显著降低。A、B、C组分别是:8.66±0.94%,8.49±0.69%,8.17±0.67%,G组是18.61±1.18%,t₁=25.62,t₂=23.59,t₃=24.17,P＜0.01;（6）治疗组（A、B、C组）与空白对照（G组）组相比较,IFN-γ表达显著增高。A、B、C组分别是:18.04±1.05%,18.56±0.82%,18.13±0.96%,G组是3.65±0.70%,t₁=44.25,t₂=53.67,t₃=47.34,P＜0.01;（7）nAChRα7、IL-4、IL-17及IFN-γ细胞因子在A组与B组之间的差别无统计学意义。nAChRα7:12.30±0.96%/12.6±1.14%,t=1.04,P＞0.05;IL-4:5.25±0.97%/4.73±0.80%,t=1.09,P＞0.05;IL-17:8.66±0.94%/8.49±0.69%,t=1.38;P＞0.05;IFN-γ:18.04±1.05%/18.56±0.82%,t=0.11,P＞0.05;（8）在CIR及各哮喘组之间,nAChRα7与IL-4表达呈正相关（r=0.76,P＜0.01）,与IL-17成正相关（r=0.46,P＜0.01）,与IFN-γ表达呈负相关（r=0.69,P＜0.01）。（9）肺组织病理改变的比较:各哮喘组（E、F、G组）及减量治疗组（D）与健康对照组（H组）相比,气道上皮增厚,管腔狭窄,管壁周围有大量炎症细胞浸润,胶原纤维增多;各治疗组（A、B、C组）经治疗后气道上皮及炎性细胞浸润情况明显改善。结论以声音或糖精水为条件刺激,以普米克令舒加万托林雾化吸入为非条件刺激建立起的条件免疫反应,可以通过下调CD4⁺T淋巴细胞上nAChRα7的表达,调节CD4⁺T淋巴细胞的免疫功能,实现对小鼠支气管哮喘的治疗。更多还原

【Abstract】 Objective（1）To observe the mechanism of the effect of conditioned immune response on mice with bronchial asthma;（2）To observe the therapeutic effect of conditioned immune response on mice with bronchial asthma;Methods An experimental asthma model was reproduced on healthy BALB/C mice（female,4-6 weeks old）sensitized and challenged with OVA.After that,all mice were divided into 7 groups randomly and treated as follows:Group A（CIR1 group）and group B（CIR2 group） established conditioned immune response by receiving the combination of conditioned stimulus（CS）and unconditioned stimulus（UCS）7times（7 days）.The former used noise（85dB）as CS and the latter used SAC.Both groups nebulised budesonide plus salbutamol as UCS.Then,they received CS once a day and the combination of CS and UCS once a week for 20 weeks;Group C（conventional therapy group）nebulized budesonide and salbutamol once a day for 20 weeks;Group D（low-dose therapy group）nebulized budesonide and salbutamol once a day for the first 7 days,and then once a week for 20 weeks;Group E（noise group）suffered noise once a day for 20 weeks;group F（SAC group）received SAC once a day for 20 weeks; Group G（blank control group）nebulized PBS once a day for 20 weeks.All the groups above were challenged with OVA once a day.Group H（healthy control group） nebulized PBS once a day for 20 weeks.After the 20-week therapy,the bronchoalveolar lavage fluid（BALF）were collected to count EOS,the spleens were collected to pick up CD4⁺ T lymphocytes,detecte the expression of alpha 7 （nAChRα7）、IL-4、IFN-γand IL-17,neuronal acetylcholine receptor,and analyze the relation among them,and the lungs were taken to observe the pathological changes by HE and MASSON staining.Results（1）After the 20-week therapy,the body weight of group D,E,F and G was much lower than that of group A,B,C and H.Meanwhile,the condition was much worse（P<0.01）（2）The percentage of EOS of group A,B and C was nuch lower than that of group G（8.67±1.23%vs 9.13±0.99%vs 8.40±1.12%vs 16.2±1.32%,t₁=16.13, t₂=16.58,t₃=17.44,P<0.01）.（3）The expression ofnAChRα7 of group A,B and C were much lower than that of group G（12.30±0.96%vs 12.6±1.14%vs 14.72±0.94%vs 15.35±1.67%, t₁=7.81,t₂=6.51,t₃=7.62,P<0.01）.（4）The expression of IL-4 of group A,B and C were much lower than that of group G（5.25±0.97%vs 4.73±0.80%vs 5.31±1.18%vs 12.26±1.31%,t₁ =16.68,t₂=19.03,t₃=15.30,P<0.01）.（5）The expression of IL-17 of group A,B and C were much lower than that of group G（8.66±0.94%vs 8.49±0.69%vs 8.17±0.67%vs 18.61±1.18%,t₁=25.62, t₂=23.59,t₃=24.17,P<0.01）.（6）The expression of IFN-γof group A,B and C were much higher than that of group G（18.04±1.05%vs 18.56±0.82%vs 18.13±0.96%vs 3.65±0.70%,t₁ =44.25,t₂=53.67,t₃=47.34,P<0.01）.（7）There was no significanly difference in the expression of neuronal acetylcholine receptors between group A and B（nAChRα7:12.30±0.96%vs 12.6±1.14%,t=1.04, P>0.05;IL-4:5.25±0.97%vs 4.73±0.80%,t=1.09,P>0.05;IL-17:8.66±0.94%vs 8.49±0.69%,t=1.38;P>0.05;IFN-γ:18.04±1.05%vs 18.56±0.82%,t=0.11, P>0.05）.（8）There was a positive correlation between nAChRα7 and IL-4（r=0.76,P< 0.01）,a positive correlation between nAChRα7 and IL-17（r=0.46,P<0.01）,and a negative correlation between nAChRα7 and IFN-γ,（r=0.69,P<0.01）.（9）The airway epithelium tissue layer of group D,E,F and G was thicker than that of group H,while the lumen were narrower and inflammatory cells and collagen fibers were much more.In contrast,the airway epithelium tissue layer and infiltration of inflammatory cells were better in group A,B and C with therapy,ConclusionsConditioned immune response,which is estabolished by receiving the combination of either noise or SAC as CS and budesonide and salbutamol as UCS, can down-regulate the expression of nAChRα7 as well as regulate the immunological functions of CD4⁺ T lymphocytes to have the same therapeutic effect on the mice with brochial asthma.更多还原