神经肽P物质诱导高糖条件下人皮肤成纤维细胞分泌MCP-1的分子机制  

Molecular mechanisms of substance P-induced monocyte chemoattractant protein-1 secretion in fibroblasts under high glucose culture condition

作　　者：贾志刚[1,2] 方勇[1,2] 姚敏[1,2] 俞为荣[1] 徐鹏[1] 贾一韬 倪涛[1,2] 

机构地区：[1]上海交通大学医学院 附属第三人民医院烧伤整形科 [2]创伤医学研究所,上海201900

出　　处：《上海交通大学学报（医学版）》2012年第10期1302-1306,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市卫生局基金(2009102);上海市交通大学医学院附属第三人民医院院基金(syz2011-13)~~

摘　　要：目的探讨神经肽P物质(SP)诱导高糖培养环境中的人皮肤成纤维细胞分泌单核细胞趋化蛋白1(MCP-1)的分子机制。方法采用高糖DMEM培养基培养人皮肤成纤维细胞,加入同一浓度(10 nmol/L)SP孵育不同时间,Western blotting检测细胞中核因子κB(NF-κB)抑制蛋白α(IκBα)、磷酸化IκBα(p-IκBα)和磷酸化NF-κB亚单位p65(p-NF-κBp65)的表达,免疫荧光方法观测细胞p-NF-κBp65表达和转位情况。将高糖DMEM培养基培养人皮肤成纤维细胞分为对照组、SP组和SP+NF-κB抑制剂(MG132)组(以MG132预处理细胞60 min),Western blotting检测各组细胞内p-NF-κBp65的表达,ELISA方法测定细胞培养上清液中MCP-1的浓度。结果随着SP作用时间的延长,成纤维细胞IκBα的表达量显著减少,p-IκBα的表达量显著增加(P<0.05);同时p-NF-κBp65的表达量在SP作用后显著增加(P<0.05),并保持递增趋势;免疫荧光染色激光共聚焦显微镜观察证实SP作用后细胞胞核内p-NF-κBp65表达增强。与SP组比较,SP+MG132组细胞p-NF-κBp65表达及细胞培养上清液中MCP-1浓度均显著降低(P<0.05)。结论 SP促进人皮肤成纤维细胞分泌MCP-1的作用机制可能与NF-κB信号通路激活有关。Objective To investigate the molecular mechanisms of substance P（SP）-induced monocyte chemoattractant protein-1（MCP-1） secretion in fibroblasts under high glucose culture condition.Methods Human skin fibroblasts were cultured in high glucose DMEM medium,and were incubated with 10 nmol/L SP for different time.The expression of inhibitor α of nuclear factor kappa B（NF-κ B）（IκBα）,phosphonated IκBα（p-IκBα） and phosphonated NF-κB subunit p65（p-NF-κBp65） in cells was detected by Western blotting,and the expression and translocation of p-NF-κBp65 was observed by immunofluorescence method.Human skin fibroblasts cultured in high glucose DMEM medium were divided into control group,SP group and SP＋NF-κ B inhibitor（MG132） group（pretreatment with MG132 for 60 min）,the expression of p-NF-κBp65 in cells was detected in each group,and the concentration of MCP-1 in the supernatant was determined by ELISA.Results The expression of IκBα significantly decreased,and that of p-IκBα significantly increased in fibroblasts with the time increase of treatment with SP（P0.05）.The expression of p-NF-κBp65 significantly increased after treatment with SP（P0.05）,which was in a time-dependent manner.The expression of p-NF-κBp65 in nuclei significantly increased after treatment with SP observed under laser confocal microscope with immunofluorescence staining.The expression of p-NF-κBp65 in cells and the concentration of MCP-1 in the supernatant in SP＋MG132 group were significantly lower than those in control group（P0.05）.Conclusion The mechanisms of SP-induced MCP-1 secretion in fibroblasts may be related to the activation of NF-κB signalling pathway.

关 键 词：P物质 人皮肤成纤维细胞 核因子κB 单核细胞趋化蛋白1 

分 类 号：R363[医药卫生—病理学]