成纤维细胞生长因子10对人角朊细胞表达GM-CSF的刺激作用

研究FGF 10对角朊细胞表达GM CSF的作用 ,以探讨FGF 10促进创面愈合的机制。按FGF 10的浓度分为 4、16、12 5和5 0 0ng/ml四组 ,细胞接种密度为低密度 (2 5 0 0细胞 /cm2 )和高密度 (5 0 0 0细胞/cm2 )两种 ,分别于FGF 10作用后 2 4、48和 72h收集细胞培养液上清并进行细胞计数 ,测定GM CSF的含量。结果显示 ,低密度接种时 ,2 4h收集的各组上清中均未能检测到GM CSF,48h上清中的12 5ng/ml和 5 0 0ng/mlFGF 10组GM CSF的浓度及单个细胞的GM CSF的分泌均显著高于阴性对照组(P <0 0 5 )。 72h的培养上清中仅5 0 0ng/mlFGF 10组GM CSF的分泌量显著高于对照组 (P<0 0 5 )。高密度接种时 ,2 4h的上清中 16、12 5、5 0 0ng/ml的FGF 10各组GM CSF浓度显著高于对照组 (P<0 0 5 ) ,但单个细胞GM CSF的分泌无显著性变化 (P >0 0 5 ) ,48h的培养上清中 ,FGF 10各组的GM CSF均未升高 ,且 48h各组单个细胞GM CSF的分泌与细胞总数呈负相关 (r2 =0 881,P <0 0 5 )。结果提示FGF 10可能通过刺激GM CSF的分泌来促进创面肉芽组织形成。

EFFECT OF FGF-10 ON THE SECRETION OF GM-CSF BY NORMAL ADULT HUMAN KERATINOCYTES IN CULTURE

To investigate the effect of FGF-10 on the secretion of GM-CSF by adult keratinocytes in vitro and to understand the mechanisms involved in the stimulation of granulation tissue formation by FGF-10 during wound healing. Concentrations of FGF-10 used were 4, 16, 125 and 500 ng/ml. Cells were seeded in the amount of 2 500 cells/cm 2 or 5 000 cells/cm 2 in dishes in serum-free medium, and supernatants were collected at 24, 48 and 72 hours after culture. The amounts of GM-CSF in cell culture supernatants were determined using GM-CSF ELISA kits, and cell numbers were counted by haemocytometer. For cells seeded in 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 cultures with 125 and 500ng/ml FGF-10 were significantly higher than that in negative control (P<0.05). At 72 hours, the production of GM-CSF in culture with 500 ng/ml FGF-10 reached an amount showing statistically significant difference 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 statistically significant difference (P>0.05). At 48 hours, the keratinocytes in the middle area were confluent, and a number of cornified cells were observed, while the productions of GM-CSF in FGF-10 cultures were not higher than that in negative control. There was a clear negative correlation between the secretion production of the growth factor and the total cell number in each dish with a correlated coeffecient of 0.881 (P<0.05). These results suggested that the effect of FGF-10 on enhancing secretion of GM-CSF by keratinocytes at certain stage might be one of the mechanisms by which FGF-10 stimulated proliferation of fibroblasts and increased formation of granulation tissue in a paracrine manner to improve wound healing.