阿维A对HaCaT细胞体外凋亡和胰岛素样生长因子结合蛋白7及血管内皮生长因子表达的影响

In vitro effects of acitretin on the apoptosis and expressions of insulin-like growth factor binding protein 7 and vascular endothelial growth factor in HaCaT cells

目的探讨阿维A对HaCaT细胞体外凋亡和胰岛素样生长因子结合蛋白7（IGFBP7）及血管内皮生长因子（VEGF）表达的影响。方法将10-5、10-6、10-7、10-8mol／L的阿维A分别作用于HaCaT细胞24、48、72h后，采用CCK8法检测细胞增殖情况；流式细胞仪检测阿维A对HaCaT细胞凋亡率的影响；Western印迹和liT—PCR法检测阿维A对HaCaT细胞IGFBP7、VEGF蛋白及其mRNA表达的影响。结果10-8mol／L阿维A处理HaCaT细胞48h时表现出对细胞增殖的抑制作用，随着时间延长和药物浓度升高，抗增殖作用亦增强，当药物浓度增加10-5mol／L时，48h和72h的抑制率分别为39．94％±2．27％和49．77％±1．87％。与对照组相比，10-5mol／L阿维A作用48h后，凋亡率由1．803％±0-313％（对照组）上升至7．617％±0．767％（阿维A组）（P〈0．05），IGFBP7的表达由0．436±0．013上升至0．939±0．040（P〈0．05），IGFBP7mRNA的表达由0．190±0．056上升至0．872±0．079（P〈0．05），VEGF的表达由0．798±0．036下降至0．213±0．032（P〈0．05），VEGFmRNA的表达由0．933±0．054下降至0．274±0．041（P〈0．05）。结论阿维A可促进HaCaT细胞的体外凋亡，并可在蛋白及mRNA水平上调IGFBP7及下调VEGF的表达。

Objective TO investigate the in vitro effects of acitretin on the apoptosis and expressions of insulin-like growth factor binding protein 7 （IGFBP7） and vascular endothelial growth factor （VEGF） in HaCaT cells. Methods Cultured HaCaT cells were treated with various concentrations （10-5, 104, 10-7, 10-8 mol/L） of acitretin for various durations, with those cultured in acitretin-free medium serving as the control group. Then, CCK- 8 assay was performed to evaluate the proliferation of cells after 24-, 48- and 72-hour treatment, flow cytometry to detect the apoptosis of HaCaT cells, and Western blot and reverse transcription-PCR to quantify the protein and mRNA expressions of IGFBP7 and VEGF in HaCaT cells, respectively, after 48-hour treatment. Statistical analysis was carried out by one-way analysis of variance and Pearson correlation analysis. Results The proliferation of HaCaT cells was inhibited by the treatment with acitretin, and the inhibitory effect increased with the elevation of concentration and prolongation of Ireatment duration of acitretin. A significant decrease was observed in the proliferative activity of HaCaT cells treated with acitretin of 104 mol/L for 48 hours, and when the concentration of acitretin was 10-5 mol/L, the proliferation of HaCaT cells was inhibited by 39.94% ± 2.27% and 49.77% ± 1.87% at 48 and 72 hours respectively, compared with the control cells. The HaCaT cells treated with acitretin of 10-5 mol/L for 48 hours showed a significant elevation in apoptosis rate （7.617% ± 0.767% vs. 1.803% ± 0.313%, P 〈 0.05）, IGFBP7 protein and mRNA expressions （0.939 ± 0.040 vs. 0.436 ± 0.013, 0.872 ± 0.079 vs. 0.190 ± 0.056, both P 〈 0.05）, but a significant reduction in VEGF protein and mRNA expressions （0.213 ±0.032 vs. 0.798 ± 0.036, 0.274 ± 0.041 vs. 0.933 ± 0.054, both P 〈 0.05） in comparison to the control cells. Conclusions Acitretin can induce the apoptosis of HaCaT cells, and up-regulate IGFBP7 but down-regulate VEGF expressions in HaCaT cells at protein and mRNA levels.