【作者】 杨丹；

【导师】 申丽；

【作者基本信息】 湖南师范大学 ， 生理学， 2017， 硕士

【摘要】 目的:研究小窝(caveolae)正常结构的维持及小窝蛋白-1(caveolin-1,cav-1)的表达对肺泡巨噬细胞分泌促炎因子IL-1β、IFN-γ及趋化因子MIP-2的影响,并探讨 CSD 肽(caveolin-1 scaffolding domain peptide)对肺泡巨噬细胞 IL-1β 表达的调节。方法:支气管肺泡灌洗分离提取Balb/c小鼠原代肺泡巨噬细胞(alveolar macrophages,AM),免疫荧光法和 Western blot 鉴定 AM 上 cav-1 的表达。FilipinⅢ(2.5μg/ml)处理RAW264.7细胞30min,ELISA测定培养液细胞因子IL-1β、IFN-γ及MIP-2的浓度。FilipinⅢ处理RAW264.7细胞30min后,再加LPS刺激6h,荧光实时定量 PCR(Real-Time PCR)测定 IL-1βmRNA 的量,ELISA 测定培养液细胞因子IL-1β、IFN-γ及MIP-2的浓度。LPS(1μg/ml)处理AM及RAW264.7 细胞 6h,Western blot 分析 caveolin-1 的表达。再用 LPS 处理 RAW264.7细胞不同时间(0.25h、0.5h、1h、2h、4h、6h),Western blot 分析 IL-lβ 的表达。不同浓度(5μmol/L、10μmol/L、20μmol/L)CSD肽预处理 AM6h后,用 LPS处理 4h;LPS 处理 RAW264.7 细胞 6h 后,加 CSD 肽(5μmol/L)处理 30h,Western blot检测IL-1β的表达。结果:1.Balb/c小鼠AM上有cav-1的表达。2.FilipinⅢ处理后RAW264.7细胞培养液细胞因子IL-1β、IFN-y及MIP-2的浓度都升高。3.LPS 处理 6h 后,AM 及 RAW264.7 细胞 caveolin-1 水平降低;且 RAW264.7细胞IL-1β mRNA表达增高,释放在培养液里的IL-1β、IFN-γ及MIP-2的蛋白水平浓度也升高;FilipinⅢ联合LPS处理会加强这些因子的表达。在LPS处理的时效实验中,IL-1β在15min和2h升高最明显。4.CSD肽预处理时,以浓度依赖方式抑制LPS刺激RAW264.7细胞生成IL-1β;CSD后处理时促进LPS刺激AM细胞生成IL-1β。结论:1.Caveolae和cav-1都参与调控炎症的发展。2.FilipinⅢ破坏巨噬细胞上caveolae的结构及LPS诱导的cav-1表达下调都会促进 IL-1β、IFN-γ 和 MIP-2 的分泌。3.CSD肽预处理能够下调LPS刺激产生的IL-1β。更多还原

【Abstract】 Objective:To investigate the possible effection of caveolae structure and cav-1 expression on IL-1β,IFN-y,MIP-2 production in alveolar macrophages and to discover the role of CSD peptide(cav-1 scaffolding domain peptide)on IL-1βexpression in alveolar macrophages.Methods:Alveolar macrophages(AM)were isolated from bronchoalveolar lavage fluid.Immunofluorescence Microscopy and Western blot were used to identify cav-1 expression in Balb/c mice AM.The concentration of IL-1β,IFN-γ and MIP-2 in culture medium of RAW264.7 cells treated with FilipinⅢ(2.5μg/ml)for 30min were measured by ELISA.The amount of IL-1β mRNA was measured by Real-Time PCR(RT PCR).The levels of IL-1β,IFN-γ and MIP-2 were measured by ELISA.Proteins of RAW264.7 cells treated with LPS(1μg/ml)for 15 min,30 min,1 h,2 h,4 h,6 h were isolated to analyze IL-1β by western blot.The possible effection of CSD peptide in different dose and treatment before or after LPS administration)on IL-1βexpression were detected by Western blot.Results:1.Cav-1 is expressed in Balb/c mice AM.2.FilipinⅢ treatment upregulated the production of pro-inflammatory cytokines IL-1β,IFN-γ and chemotactic factor MIP-2 in RAW264.7 cells(P<0.05).3.LPS-challenge decreased cav-1 and increased IL-1β,IFN-y and MIP-2(P<0.05).The present of FilipinⅢ enhanced upregulation of these three cytokines induced by LPS(P<0.05).4.CSD peptide pretreatment for 6 hours attenuated the IL-1β production induced by LPS,while 30-hour-treatment with CSD peptide increased the IL-1β production induced by LPS(P<0.05).Conclusions:1.Caveolae and cav-1 help to regulate inflammation development.2.The caveolae structure and cav-1 expression could modulate the production of IL-1β,IFN-γ and MIP-2 in macrophages.3.CSD peptide treatment in right time could attenuate the IL-1β production induced by LPS.更多还原