UVB应激性衰老成纤维细胞分泌细胞外基质对HaCaT细胞ERK信号的影响

Influences of extracellular mat rices secreted by ultraviolet B-induced senescent fibroblasts on ERK signaling in HaCaT cells

目的探讨UVB诱导的衰老（UVB—SIPS）成纤维细胞分泌的细胞外基质（ECM）对HaCaT细胞增殖的影响以及细胞外信号调节激酶（ERK）信号途径的作用。方法采用辐射诱导人皮肤成纤维细胞衰老，分别制备由未衰老和衰老成纤维细胞分泌的ECM包被的培养皿（分别定为PRE—ECM组、SCIP—ECM组）。以空白培养皿（NON—ECM组）为参照，在HaCaT细胞接种于培养皿后，采用MTF法和流式细胞法检测其细胞增殖、细胞周期等指标的变化；免疫印迹法分析ERK活化水平。干预性研究ERK信号通路对上述功能的影响。结果三组中，SCIP—ECM组可诱导最强烈的ERK1／2的活化。采用U0126抑制ERK信号活化可完全抑制衰老成纤维细胞来源的ECM的促细胞增殖效应。阻断HaCaT细胞的ERK活化，NON—ECM组、PRE—ECM组和SCIP—ECM组S＋G2／M期细胞的比例明显下降，分别由处理前37．40％、41．34％和43．31％减少至29．41％、36．48％到39．96％。结论UVB—SCIP成纤维细胞分泌的ECM通过刺激ERK磷酸化促进HaCaT细胞增殖。

Objective To explore the influences of extracellular matrices （ECM） secreted by ultraviolet B （UVB）-induced senescent fibroblasts on the proliferation of and extracellular signal-regulated kinase （ERK） signaling in HaCaT cells. Methods Fibroblasts were irradiated with UVB of 15 mJ/cm^2 once daily for 5 days to induce premature senescence, which was identified by SA-13-gal staining 72 hours after the last irradiation. HaCaT cells were divided into 3 groups and inoculated into plates coated with extracellular matrices secreted by non-senescent （PRE-ECM） or senescent fibroblasts （SIPS-ECM） or into uncoated plates （NON-ECM）, followed by additional culture. U0126, an inhibitor of ERK1/2, was used to treat the HaCaT cells 1 hour before inoculation. Then, MTT assay was carried out to detect the proliferation of HaCaT cells after a 3-day culture, Western blot to assess the phosphorylation of ERK at 0.5, 1, 2 and 4 hours after the inoculation, flow cytometry to analyse cell cycle and apoptosis after 24 hours of culture. Results The most rapid and intense phosphorylation of ERK was observed in SIPS-ECM group. Inhibiting the activation of ERK pathway with U0126 could completely suppress the promoting effect of ECM from senescent fibroblasts on the proliferation of HaCaT cells. After the blocking of ERK activation, the proportion of HaCaT cells in S and G2/M phase decreased from 37.40%, 41.34% and 43.31% to 29.41%, 36.48% and 39.96%, respectively, in NON-ECM, PRE-ECM and SCIP-ECM group. Conclusion The ECM produced by UVB-induced senescent fibroblasts promote the proliferation of HaCaT cells via inducing the phosphorylation of ERK.