感觉神经肽P物质对肉芽组织成纤维细胞表达表皮生长因子与成纤维细胞生长因子-2及受体的调控作用

Sensory neuropeptide SP modulating expression of EGF,FGF-2 and their receptors in fibroblasts of granulation in vivo and in vitro

目的 从基因水平探讨感觉神经肽SP对肉芽组织成纤维细胞表达表皮生长因子(EGF)、成纤维细胞生长因子 2 (FGF 2 )及受体的调控作用。方法 由在体和离体实验两部分组成。在体实验采用Wistar大鼠 5 0只 ,随机分为辣椒素组和对照组。辣椒素组大鼠皮下注射辣椒素 5 0mg/kg毁损感觉神经 ,1周后行皮肤全层切割伤 ,采用免疫组化结合图像分析 ,观察伤后 3～ 12d伤口肉芽组织内神经肽P物质 (SP)含量。采用原位杂交方法观察EGF、表皮生长因子受体 (EGFR)、FGF 2、成纤维细胞生长因子受体 1(FGFR 1)的基因表达 ,对照组除不化学毁损感觉神经外 ,余处理同辣椒素组。离体实验采用RT PCR技术观察不同浓度 (10 -9～ 10 -5mol/L)SP作用于培养肉芽组织成纤维细胞后 ,上述生长因子及受体的表达。结果 在体实验示 ,肉芽组织SP免疫阳性染色与EGF、FGF 2及其受体表达有关 ,化学毁损感觉神经 ,肉芽组织SP免疫阳性染色减弱 ,上述因子及受体的表达也明显抑制。离体实验发现 ,上调培养肉芽组织成纤维细胞内源性EGF和EGFRmRNA表达的SP浓度为 10 -8～10 -6mol/L和 10 -6～ 10 -5mol/L ,上调FGF 2和FGFR 1mRNA表达的SP浓度分别为 10 -9～ 10 -5mol/L和 10 -6～ 10 -5mol/L。结论 伤口愈合中 ,感觉神经释放的SP参与肉芽组织成纤维?

Objective To study the modulating effect of sensory neuropeptide substance P (SP) on gene expression of epidermal growth factor(EGF), fibroblast growth factor-2(FGF-2) and EGF receptor(EGFR), FGF receptor-1(FGFR-1) in granulation fibroblast. Methods The study consisted of two parts, in vivo and in vitro. In vivo, fifty wistar rats were randomly arranged into capsaicin group and control group. The rats in capsaicin group were subcutaneously injected with neurotoxin dose of capsaicin (50 mg/kg) on the back of the rats to chemically destroy sensory nerves to prevent the release of SP. After one week, full-thickness skin wounds were created on the rats′ back .SP content and EGF, EGFR, FGF-2, FGFR-1 gene expression in the granulation tissue were observed using immunohistochemistry and in situ hybridization combined with image analysis on 3 rd, 6 th, 9 th 12 th after skin injury, respectively . The experimental procedure in control group were similar with that in capsaicin group except injection with capsaicin. In vitro, SP (10 -9 -10 -5 mol/L) was added into culture medium for cultured fibroblasts from granulation and mRNA expressions of EGF, FGF-2 and their receptors were assayed with RT-PCR. Results In the present study, immunoreactive stain for SP in granulation tissue were correlated with gene expression of EGF,FGF-2 and their receptors using in situ hybridization in vivo. Once sensory nerves were destroyed and SP release was inhibited in capsaicin group, the gene expression of above growth factors and their receptors were attenuated. SP concentrations in culture medium for upregulation of mRNA expressions of EGF and EGFR in cultured fibroblasts were from 10 -8 mol/L to 10 -6 mol/L and from 10 -6 mol/L to 10 -5 mol/L, respectively. SP concentration was from 10 -9 mol/L to 10 -5 mol/L for upregulation of FGF-2 mRNA expression in cultured fibroblasts and from 10 -6 mol/L to 10 -5 mol/L for upregulation of FGFR-1. Conclusion Sensory neuropeptide SP released from sensory nerve participates in upregulation of gene expressions of EGF, EGF-2 and their receptors in granulation fibroblasts during wound healing.