IL-31在慢性支气管哮喘中的表达及其作用机制研究

IL-31 is thought to potentially mediate interactions between.T cells and epithelial cells. High level of Interleukin-31 is detected in peripheral blood of patients with allergic asthma, and IL-31 receptor was upregulated in lung tissue and bronchial epithelial cells of mouse model of allergic asthma. Interleukin-31 may be involved in airway inflammation by inducing production of chemokines such as MCP-1, IL-8 from bronchial epithelial cells. In this study, We will established mouse model of chronic alergic asthma with OVA to investigate the expression of IL-31 and IL-31 receptor in lung tissue. The expression of chemokines in lung tissue and bronchus will be detected by cDNA microarrays. According to cDNA microarrays, some chemokines will be selected out to stimulate macrophages and T cells. The chemotaxis of chemokines in vitro and airway inflammation in lung tissue will be analysed. Rabbits and BALb/c mice will be injected with IL-31 protein produced in E.coli under induction of IPTG to prepare polyclonal antibodies and monoclonal antibodies respectively, then IL-31 ELISA detecting kit will be manufactured. The expression of IL-31 in peripheral blood sample from patiets with chronic allergic asthma will be analysed by enzyme-linked immunosorbent assay, in stage of asthmatic attack and paracmasis. The mechnism of interleukin-31 and the relationship between IL-31 and asthmatic attack will be investigated. This study may help us to understand important roles of IL-31 in the development of asthmatic attack, and suggust novel pharmacological targets for future drug development.

支气管哮喘患者外周血IL-31表达增加，哮喘小鼠模型支气管脱落细胞IL-31受体表达增高，IL-31体外可刺激人支气管上皮细胞表达趋化因子MCP-1、IL-8等，IL-31参与哮喘气道炎症，作用机制可能和IL-31诱导肺组织表达多种趋化因子相关。本项目在前期研究IL-31促炎作用的基础上，建立慢性哮喘小鼠模型，观察小鼠肺组织炎症细胞浸润，基因芯片技术检测肺组织趋化因子表达谱；分析IL-31和IL-31受体在肺组织中的表达和细胞定位，体外研究IL-31对支气管、肺组织细胞趋化因子表达谱的影响；根据趋化因子的表达，研究相关趋化因子对巨噬细胞、T细胞的趋化效应及细胞募集到肺组织后的炎性损伤效应，并检测IL-31在支气管哮喘病人外周血中的表达，探讨IL-31的作用机制及其与支气管哮喘病人哮喘发作的关系。项目的预期结果将有助于支气管哮喘气道炎症发生发展机制的完善，为哮喘病人的治疗提供依据。

支气管哮喘患者外周血IL-31及哮喘小鼠模型支气管脱落细胞IL-31受体表达增高，IL-31体外可刺激人支气管上皮细胞表达趋化因子MCP-1、IL-8等，表明IL-31参与哮喘气道炎症，作用机制可能和IL-31诱导肺组织表达多种趋化因子相关。本项目在前期研究IL-31促炎作用的基础上，采用常规OVA法建立急性及慢性支气管哮喘小鼠模型，ELISA及荧光定量PCR动态分析见哮喘小鼠外周血及肺泡灌洗液IL-31水平增高，肺组织IL-31受体表达增加。为了探讨IL-31在哮喘气道炎症中的作用，体外分离培养小鼠Ⅱ型肺泡上皮细胞，IL-31作用后经基因芯片分析细胞多种趋化因子及其受体的表达增高，IL-31作用后的细胞培养上清对巨噬细胞、T淋巴细胞有趋化作用；体内趋化实验发现，CFSE标记的巨噬细胞经尾静脉注射后可被趋化到哮喘小鼠肺组织。分析人IL-31抗原表位，用合成免疫原BSA-IL-31免疫Balb/c小鼠，取小鼠脾细胞与骨髓瘤细胞进行融合，对阳性克隆进行筛选和亚克隆，经ELISA鉴定成功获得了2株表达人IL-31单克隆抗体的杂交瘤细胞，杂交瘤细胞腹腔注射小鼠，获得了高效价的特异性人IL-31单克隆抗体。用HRP标记IL-31抗体，成功建立了IL-31 ELISA检测体系，自制试剂盒特异性好，与IL-1、IL-6等无交叉反应。采用自制的IL-31检测试剂盒对19例哮喘病人血清进行分析，结果见哮喘病人血清IL-31水平明显高于健康对照。为了深入研究IL-31在哮喘气道炎症中的作用机制，课题组在本项目原计划的基础上增加了新的内容：1、建立了IL-31RA基因敲出小鼠模型，初步研究了IL-31受体信号在哮喘气道炎症中的作用及IL-31信号对T淋巴细胞增殖及分化的影响；2、采用杂交瘤技术，成功获得了分泌小鼠IL-31单克隆抗体的杂交瘤细胞，为采用IL-31单克隆抗体阻断研究IL-31作用及机制奠定了基础。