落新妇苷对永生化人角质形成细胞 HaCaT增殖、凋亡的影响及机制探讨

摘要

目的：观察土茯苓提取物落新妇苷对永生化人角质形成细胞HaCaT增殖、凋亡的影响，并探讨其机制。方法收集对数生长期HaCaT细胞，观察组分别加入不同浓度落新妇苷、空白对照组加DMEM培养液，培养24 h后，采用MTT法测算细胞增殖抑制率，流式细胞仪检测细胞早期凋亡率；收集对数生长期HaCaT细胞，观察组分别加入不同浓度落新妇苷＋20 ng／mL INF-γ、模型对照组加20 ng／mL INF-γ、空白对照组加DMEM培养液培养24 h，采用实时荧光定量 PCR 法检测细胞中的核转录因子κB （ NF-κB ） p65、骨桥蛋白（ OPN ）、血管内皮生长因子（VEGF） mRNA。结果落新妇苷在25、50、100、200、400μg／mL 时，HaCaT 细胞增殖抑制率分别为3．06％、26．53％、40．82％、60．20％及61．22％。落新妇苷在50、100、200μg ／mL时，观察组HaCaT细胞早期凋亡率分别为3．26％、8．24％和15．38％；与空白对照组早期凋亡率（1．33％）比较，P均＜0．05。与空白对照组比较，模型对照组NF-κB p65、OPN、VEGF mRNA表达增加（P均＜0．01）；与模型对照组比较，观察组随落新妇苷浓度增加，NF-κB p65、OPN、VEGF mRNA表达逐渐降低（P均＜0．05）。结论土茯苓提取物落新妇苷能抑制HaCaT细胞增殖、诱导细胞凋亡，其机制可能与制抑细胞NF-κB p65、OPN、VEGF mRNA表达有关。%Objective To observe the effects of astilbin on the cell proliferation and apoptosis of human keratinocyte cell line HaCaT and to investigate the mechanism .Methods HaCaT cells in the logarithmic phase were selected .The ob-servation group was incubated with different concentrations of astilbin , and the blank control group was cultured with DMEM solution for 24 hours.The proliferation and apoptosis of HaCaT cells were respectively measured by MTT and flow cytometry (FCM).HaCaT cells in the logarithmic phase were selected .The observation group was cultured with different concentra-tions of astilbin+20 ng/mL INF-γ, the model group was cultured with 20 ng/mL INF-γand the blank control group was cultured with DMEM solution for 24 hours.Real-time fluorogenetic quantitative PCR (FQ-PCR) was employed to detect the expression of nuclear transcription factor-κB p65 (NF-κB p65), osteopontin (OPN) and vascular endothelial growth factor (VEGF) mRNA.Results When the concentrations of astilbin were 25, 50, 100, 200, 400 μg/mL, the proliferation in-hibition rates of HaCaT cells were 3.06%, 26.53%, 40.82%, 26.53%and 61.22%.When the concentrations of astil-bin were 50, 100, 200μg/mL, the early apoptosis rates of HaCaT cells in the observation group were 3.26%, 8.24%and 3.26%, respectively.Compared with the blank control group (1.33%), significant difference was found in the early ap-optosis rate (all P<0.05).Compared with the blank control group , the expression of NF-κB p65, OPN and VEGF mRNA was increased in the model control group (all P<0.01).Compared with the model control group , the expression of NF-κB p65, OPN and VEGF mRNA was gradually decreased with the increased concentrations of astilbin (all P<0.05).Con-clusion Astilbin can inhibit the proliferation and induce the apoptosis of HaCaT cells , whose mechanism may be associat-ed with the inhibition of VEGF , NF-κB p65 and OPN mRNA expression .