【作者】 杨平满；

【导师】 周建英；

【作者基本信息】 浙江大学 ， 内科学， 2009， 硕士

【摘要】 背景和目的支气管哮喘简称哮喘,是世界范围内严重危害公众健康的一种常见病,其病因及发病机制复杂、治疗棘手,难以根除。糖皮质激素(GCs)是治疗哮喘的有效药物,已能有效控制大多数患者的症状,但长期应用可带来严重的副作用。最近发达国家的哮喘普遍增加并成为主要的健康和经济后果令人不安。寻找安全有效的哮喘治疗切入点十分迫切。Th2型细胞源性细胞因子的中心作用表明,治愈或提供长期解除哮喘症状需针对变应原特异性Th2细胞反应。哮喘患者体内存在IL-10含量下降及调节性T细胞(Tregs)功能受损。IL-10的分泌Tregs以IL-10依赖方式抑制变应原特异性Th2细胞细胞因子分泌。一种替代和更具吸引力的策略是通过诱导具有提供安全和长期减轻疾病症状潜力的变应原激活的IL-10的分泌Tregs提供内源性抑制信号。诱导抗原特异性IL-10的分泌Tregs是今后哮喘治疗一个有吸引力的领域。IL-10在IL-10的分泌Tregs的功能活动及抑制Th2反应和过敏反应中发挥重要作用,其强力的抗炎和抑制免疫性质成为一个控制哮喘反应的有吸引力的细胞因子。研究表明,某些免疫方案及钙通道阻滞剂能诱导IL-10的合成,临床上对GCs没有反应的病人在体内同样不能出现GCs诱导的IL-10的合成,诱导IL-10的合成可能有助于GCs治疗哮喘的临床疗效。有可能利用这些影响,通过GCs单独或连同其他药物治疗诱发抗原特异性IL-10的分泌Tregs,诱导IL-10的合成改善哮喘症状。环孢霉素A(CsA)是一个众所周知的通过抑制钙调神经磷酸酶(CaN)活性阻止核因子NF-AT移位到细胞核的免疫抑制剂,能抑制哮喘气道炎症,减少哮喘GCs用量,但其具体作用机制尚未阐明。本研究旨在观察CsA对卵白蛋白(OVA)诱导的小鼠哮喘模型细胞因子IL-10、IL-4、IL-5、IL-2、IFN-γ及炎症细胞、CD4~+CD25~+IL-10~+T细胞的影响,将IL-10、CD4~+CD25~+IL-10~+T细胞联系起来进一步探讨CsA抑制哮喘炎症、免疫的可能机制,为临床综合治疗及开发新药提供理论及实验依据。方法实验分为对照组,哮喘模型组,CsA组和CsA+IL-10抗体组。用OVA腹腔致敏、雾化激发建立哮喘BALB/c小鼠模型(n=40)。ELISA法检测BALF中IL-4、IL-5、IL-2、IL-10和IFN-γ水平。肺组织病理切片HE、AB/PAS染色观察肺组织炎症情况。流式细胞仪检测肺组织CD4~+CD25~+IL-10~+T细胞百分率。结果CsA雾化后BALF中炎症细胞细胞数量明显减少,IL-4、IL-5、IL-2水平明显降低(p<0.01),IL-10的含量明显增高(p<0.01)。切片HE、AB/PAS染色显示肺组织及杯状细胞炎症消退明显。同时,肺组织CD4~+CD25~+IL-10~+T细胞百分率明显高于对照组及模型组(p<0.01)。使用IL-10抗体后哮喘炎症较单独使用CsA组加重。结论1 CsA可以抑制哮喘小鼠气道炎症,该项作用可能通过上调IL-10分泌来实现,CD4~+CD25~+IL-10~+T细胞可能在其中起重要作用。2 CsA通过诱发CD4~+CD25~+IL-10~+T细胞,诱导IL-10的合成可能有治疗哮喘的潜在理论和实践价值。更多还原

【Abstract】 Background and Purpose:Bronchial asthma,also named asthma,is a common disease which becomes a severe hazard to public health,affected worldwide.As a result of the unknown etiology and complex mechanism,the therapy of asthma is still an intractable problem.Although inhaled glucocorticoids(GCs) are very effective for the treatment of most patients with asthma,an important subgroup of patients require oral GCs to control their disease at the risk of considerable side effects.The recent disturbing increase in the prevalence of asthma in the developed world has major health and economic consequences.Thus,it is imperative that we should look for a breakthrough in the asthmatic treatment.The central role of Th2-cell-derived cytokines indicates that strategies to cure or provide long-term relief from asthma symptoms need to target this allergen-specific Th2 response.The patients of asthma usually have an impair function of regulatory T cells(Tregs) and a low level of IL-10.allergen-induced cytokine production by allergen-specific Th2 cells was inhibited by allergen-induced IL-10-secreting Tregs in an IL-10-dependent manner.an alternative and more attractive strategy is the local delivery of inhibitory signals through the induction of allergen-activated IL-10-secreting Tregs.Induction of antigen-specific IL-10-secreting Tregs is an appealing future therapeutic area for asthma.IL-10 plays an essential role in the functional activities of IL-10-secreting Tregs and in the regulation of Th2 responses and allergic responses.The anti-inflammatory and immunological properties of IL-10 make it an attractive cytokine for the control of asthmatic responses.Several immunomodulatory regimens and calcium channels blocker have been shown to increase IL-10 synthesis.Induction of IL-10 synthesis may contribute to the clinical efficacy of GCs therapy in asthma since patients who fail to respond clinically to GCs also fail to respond ex vivo to GCs for induction of IL-10 synthesis.it might be possible to harness these effects to promote the induction of antigen-specific IL-10- secreting Tregs,either by treatment with GCs alone or together with other drugs,induction of IL-10 synthesis is associated with amelioration of disease symptoms.Cyclosporine A(CsA) is a well-known immunosuppressant that blocks NF-AT translocation into the nucleus by inhibition calcineurin phosphatease activity.It was proved that can inhibit airway inflammation and decrease the dosage of GCs,but the mechanism of its anti-inflammatory function is still unclear.The purpose of our study is to observe the influence of CsA on the level of IL-10、IL-4、IL-5、IL-2、IFN-γand inflammatory cells in ovalbumin(OVA) -induced mouse model and detect the relationship between IL-10 and CD4~+CD25~+IL-10~+T cells,consequently,investigate the mechanism of resolution in asthmatic inflammation and provide the oretical basis for the clinical therapy and development of new drugs in asthma.Material and methods:BALB/c mice(n=40) were divide into four groups:control group,model group,CsA group and CsA +anti-IL-10 antibody group(n=40).Mice were sensitized to OVA by intraperitoneal injection and challenged with OVA aerosol for 7 consecutive days.The concentration of cytokines including IL-4、IL-5、IL-2、IL-10 and IFN-γin BALF were measured by using ELISA Kits.Lung tissue sections were stained with hematoxylin-eosin(HE) and Alcian blue /periodic acid Schiff(AB/PAS) to observe general morphology and goblet cells.Flow cytometry were used to detect the percentage of CD4~+CD25~+IL-10~+T cells. Results:Treated by CsA aerosol significantly reduced the numbers of inflammatory cells and the level of IL-4JL-5 and IL-2,also,resolved pulmonary infiltration of inflammatory and goblet cells hyperplasia,with a high level of IL-10 in BALF (p<0.01).Further,the percentage of CD4~+CD25~+IL-10~+T cells presented negative correlations with the numbers of inflammatory cells,goblet cells hyperplasia and the levels of Th2 cytokines(p<0.01).Conclusions:1.CsA can inhibit airway inflammation in asthmatic mouse,as a result of the up-regulation in the level of IL-10 probably.CD4+CD25+IL-10+T cells may play an important role during the process.2.CsA-induced increase of CD4~+CD25~+IL-10~+ T cells to up-regulate the level of IL-10 may provide theoretical basis and practical value.更多还原