成纤维细胞生长因子-10刺激人角朊细胞表达粒细胞-巨噬细胞集落刺激因子

Effect of fibroblast growth factor-10 on the secretion of granalocyte-macrophage colony stimulating factor by normal adult human keratinocytes in vitro

目的:探讨成纤维细胞生长因子-10(FGF-10)促创面肉芽组织形成的作用机制。方法:将不同浓度的 FGF-10分别加入细胞培养液中,于作用后24、48和7,2h 收集细胞培养上清,采用 ELISA 方法测定粒细胞—巨噬细胞集落刺激因子 GM-CSF 的含量,同时进行细胞计数。结果:当细胞接种密度为2 500细胞/cm^2时,24h 的培养上清中未能检测到 GM-CSF,48h 的上清中125 ng/ml 和500ng/ml FGF-10组 GM-CSF 的浓度及单个细胞的 GM-CSF 的分泌均显著高于对照组(P<0.05)。72h的培养上清中仅500ng/ml FGF-10组 GM-CSF 的分泌量显著高于对照组(P<0.05)。当细胞接种密度为5 000细胞/cm^2时,16～500ng/ml 的 FGF-10各组 GM-CSF 浓度显著高于对照组(P<0.05),但单个细胞的 GM-CSF 分泌量与对照组无显著差异(P>0.05),48h 收集的培养上清中,与对照组相比,FGF-10各组的 GM-CSF 均未升高,且48h 各组单个细胞的GM-CSF 分泌量与该培养皿中的细胞总数呈负相关(r=-0.881,P<0.05)。结论:实验结果提示 FGF-10可能通过刺激 GM-CSF 的分泌,从而间接作用于成纤维细胞、内皮细胞等,促进创面肉芽组织形成。

Objective:To investigate the effect of fibroblast growth factor-10(FGF-10)on the secretion of granu- locyte-macrophage colony stimulating factor (GM-CSF)by adult keratinocytes in vitro and its potential mecha- nisms of stimulation of granulation tissue formation by FGF-10 during wound healing.Methods:Concentrations of FGF-10 used were 4,16,125 and 500 ng/ml.Cells were seeded at 2 500 eells/cm^2 or 5 000 cells/cm^2 in dishes in serum-free medium and supernatants were collected at 24,48 and 72 hours.The amounts of GM-CSF in cell cul- ture supernatants were determined using GM-CSF ELISA kits,and cell numbers were counted by haemocytome- ter.Results:For cells seeded at low density (2 500 cells/cm^2 ),GM-CSF was not detected at 24 hours.At 48 hours,both in absolute concentrations and on a per-cell basis,the amounts of GM-CSF secreted in 125 and 500 ng/ml FGF-10 were both significantly higher than that in negative control (P<0.05).At 72 hours,the produc- tion of GM-CSF in 500 ng/ml FGF-10 reached statistical significance compared with negative control (P<0.05). For cells seeded at 5 000 cells/cm^2 and cultured in KGM-2 without EGF for 72 hours,the absolute concentrations of GM-CSF in 16-500 ng/ml FGF-10 were significantly higher than that in negative control at 24 hours (P< 0.05),however,the secretion per cell did not reach statistical significance (P>0.05).At 48 hours,the keratino- cytes in the middle area were confluent and a number of cornified cells were observed,and the productions of GM- CSF in FGF-10 were not higher than that in negative control.There was a clear negative correlation between the secreted production of the growth factor and the total cell number in each dish with a correlated coeffecient of 0.881(P<0.05).Conclusions:These results suggested that the effect of FGF-10 on increasing secretion of GM- CSF by keratinocytes at certain stage might be one of the mechanisms by which FGF-10 stimulates proliferation of fibroblasts and increases formation of granulation tissue in a paracrine manner to improve wound healing.