光老化成纤维细胞分泌的细胞外基质对HaCAT细胞增殖影响及FAK/PI3-K/Akt信号途径的作用机制研究

Effect of the ECM secreted from photoaging fibroblasts on proliferation of HaCAT cells and mechanism mediated by FAK/PI3-K/Akt signaling

目的探寻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FAK和PI3-K/Akt信号途径在其中所起的作用。方法连续亚细胞毒剂量UVB辐照诱导皮肤成纤维细胞衰老,制备由衰老成纤维细胞分泌的ECM,接种HaCAT细胞至ECM包被的平皿,观察空白ECM、未衰老的ECM和光老化的ECM对HaCAT增殖、凋亡等功能的影响。免疫印迹法检测细胞内FAK和Akt磷酸化水平的时相性变化。在干预研究中,采用细胞松弛素D和渥曼青霉素阻断FAK和PI3-K/Akt信号通路,对比观察对细胞功能和信号的影响。结果①3组均可见Akt轻度但无显著差异的磷酸化的增加;但U-VB-SIPS组的FAK信号发生最快最明显的增加。②2μM细胞松弛素D可明显抑制FAK和Akt磷酸化;同时,细胞的凋亡率也显著增加。③以400nM渥曼青霉素预处理细胞,明显抑制Akt磷酸化,同时3组的凋亡水平分别达15.8%、23.4%和21.8%。④细胞松弛素D和渥曼青霉素均明显抑制了光老化的ECM促细胞增殖效应。结论在本系统中FAK/PI3-K/Akt信号途径发挥抑制细胞凋亡的作用,阻断该信号通路可完全破坏UVB-SIPS成纤维细胞分泌的细胞外基质对HaCAT的促增殖作用。

【Objectives】To explore the mechanism that the extracellular matrices （ECM） secreted by UVB-stress induced premature senescent （UVB-SIPS） fibroblast promoting the proliferation of HaCAT cells and how FAK/PI3K/Akt signaling pathway contribute to such effects.【Methods】Presenescent human skin fibroblasts （HSF） were exposed to repeated subcytotoxic UVB until the HSF developed senescent response.The ECM secreted by presenescent and HSF in UVB-SCIP were prepared.HaCAT cells were plated onto them.The rate of HaCAT cells apoptosis and cell-cycle progression was detected with Flow Cytometry.The proliferation of HaCAT was measured with MTT assay.The level of phosphorylation of FAK and Akt was measured by western blot method in a time-dependent manner.Cytochalasin D and wortmaninn were used to block FAK and PI3-K/Akt,respectively.【Results】①The attachment of HaCAT to non-ECM and ECM deposited by HSFs in UVB-SIPS or by presenescent HSFs inducing a slight but not significant phosphorylation of Akt,but the ECM deposited by HSFs in UVB-SIPS could induce the most rapid and intense phosphorylation of FAK.②The pretreatment with 2 μM cytochalasin D in HaCAT inhibited phosphorylation of FAK and Akt.At the same time,the rate of cells apoptosis increased significantly.③The pretreatment with 400 nM wortmannin inhibited the phosphorylation of Akt,and dramatically enhanced the proportion of apoptotic cells among all of three groups by 15.8%,23.4% and 21.8%,respectively.④Both cytochalasin D and wortmannin could inhibit the proliferation of HaCAT cells mediated by the ECM secreted from HSF in UVB-SIPS.【Conclusion】FAK/PI3-K/Akt works as a protector of HaCAT cells from apoptosis in our system,the inhibition of which would overwhelm the proliferation effect induced by ECM deposited by HSFs in UVB-SIPS.