P物质对高糖条件下成纤维细胞增殖及其单核细胞趋化蛋白-1合成的调节作用

摘要

目的 探讨外源性P物质(substance P,SP)对高糖条件下人皮肤成纤维细胞(human skin fibroblast,HSF)增殖及其单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)合成的影响. 方法 用高糖DMEM培养基培养HSF；并用SP在不同浓度和不同时相点处理,ELISA法检测细胞上清液中MCP-1分泌水平,观察时问与剂量的时效关系；半定量RT-PCR、实时荧光定量(Real Time)-PCR、Western blot分别检测各组HSF内MCP-1 mRNA的表达和蛋白的合成；细胞增殖试剂盒(cell counting Kit-8,CCK-8)检测不同浓度SP作用HSF后的增殖情况. 结果 经SP处理后8h可检测到MCP-1分泌增强(P＜0.05),24h达峰值(P＜0.01),在10,100 nmol/L的浓度下分泌最多(P＜0.01)；SP可明显增加高糖条件下HSF MCP-1mRNA的表达和蛋白的合成,特别是在10 nmol/L的浓度下表达最为显著(P＜0.01)；在10,1 000nmol/L的SP均明显促进HSF的增殖(P＜0.05). 结论 SP可促进高糖条件下HSF的增殖及MCP-1的合成,对促进糖尿病创面修复可能有一定意义.%Objective To investigate effects of exogenous substance P (SP) on proliferation of humau skin fibroblast (HSF) and expression of its monocyte chemoattractant protein-1 (MCP-1) under high glucose condition.Methods HSF cultured in high glucose DMEM medium were treated with SP of various concentrations at different time points.Levels of MCP-1 in the supernatants were determined by ELISA and time-effect relationship of SP regulating expression of MCP-1 was analyzed.Expression of MCP-1 mRNA in HSF was detected by semi-quantitative RT-PCR and Real Time-PCR,Conclusion,while cxpression of MCP-1 by Western blot.Proliferation of HSF treated with SP of different concentrations was detected by cell counting Kit-8 (CCK-8).Results MCP-1 had a strong expression at 8 hours after SP disposal (P ＜0.05) and reached the peak at 24 hours (P ＜ 0.01).Meanwhile,MCP-1 expressed the most at 10 nmol/L and 100 nmol/L SP (P ＜0.01).SP significantly enhanced the expression of MCP-1 mRNA and its synthesis under high glucose condition,especially at the concentration of 10 nmol/L (P ＜0.01).Also,SP induced obvious proliferation of HSF at concentrations of 10 nmol/L and 1 000 nmol/L(P ＜ 0.05).Conclusion SP promotes proliferation of HSF and expression of MCP-1 in high glucose DMEM medium,which may be of significance in promoting diabetic wound healing.