上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (10): 1302-.doi: 10.3969/j.issn.1674-8115.2012.10.005

• 论著（基础研究） • 上一篇    下一篇

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

贾志刚1,2, 方 勇1,2, 姚 敏1,2, 俞为荣1, 徐 鹏1, 贾一韬2, 倪 涛1,2   

  1. 上海交通大学 医学院 1.附属第三人民医院烧伤整形科, 2.创伤医学研究所, 上海 201900
  • 出版日期:2012-10-28 发布日期:2012-11-05
  • 通讯作者: 倪 涛, 电子信箱: nitao6666@yeah.net。
  • 作者简介:贾志刚（1983—）, 男, 硕士生；电子信箱: jiazhigang.5@163.com。
  • 基金资助:

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

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

JIA Zhi-gang1,2, FANG Yong1,2, YAO Min1,2, YU Wei-rong1, XU Peng1, JIA Yi-tao2, NI Tao1,2   

  1. 1.Department of Burns and Plastic Surgery, the Third People´s Hospital, 2.Institute of Traumatic Medicine, Shanghai Jiaotong University School of Medicine, Shanghai 201900, China
  • Online:2012-10-28 Published:2012-11-05
  • Supported by:

    Shanghai Municipal Health Bureau Foundation, 2009102；Foundation of the Third People´s Hospital, Shanghai Jiaotong University School of Medicine, syz2011-13

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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-κ B）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。

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

Abstract:

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-κB)(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-κB 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 (P<0.05). The expression of p-NF-κBp65 significantly increased after treatment with SP (P<0.05), 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 (P<0.05). Conclusion The mechanisms of SP-induced MCP-1 secretion in fibroblasts may be related to the activation of NF-κB signalling pathway.

Key words: substance P, human skin fibroblasts, nuclear factor kappa B, monocyte chemoattractant protein-1