摘要目的 探讨窄谱中波紫外线(NB-UVB)对HaCaT细胞表达Gadd45α及对细胞增殖、周期的影响.方法 分别以100、200、400 mJ/cm2NB-UVB照射HaCaT细胞后,于6、12、24 h收集细胞,采用RTPCR和Western印迹检测Gadd45α mRNA和蛋白水平的变化;CCK8法检测照射后对HaCaT细胞增殖的影响;流式细胞术检测照射后对HaCaT细胞周期的影响.结果 HaCaT细胞表达Gadd45α,分别经100、200、400 mJ/cm2NB-UVB照射后,Gadd45α mRNA及蛋白表达水平均在6 h升高,12 h升高明显,24 h表达下降,与未照射组比较差异均有统计学意义(P<0.05).三个剂量组照射后12 h,Gadd45α mRNA相对A值分别为1.4360±0.6551、1.8633±0.0979、1.9266±0.1724,均高于对照组(0.6000±0.1276)(P<0.05);Gadd45α蛋白A值分别为0.0773±0.0005、0.1936±0.0015、0.2373±0.0015,较对照组(0.0290±0.0010)增高(P<0.05).NB-UVB照射对HaCaT细胞有抑制作用,其抑制增殖作用呈量效及时效关系;三组照射后HaCaT细胞周期发生改变,G:期细胞比例依次为13.53±1.03、17.77±2.25、30.03±4.29,较对照组(9.24±0.97)显著增高(P<0.05),细胞周期被阻滞于G2期.结论 NB-UVB照射后HaCaT细胞上调Gadd45α表达;Gadd45α可能参与了紫外线照射后HaCaT细胞增殖抑制、周期阻滞过程.

abstractsObjective To investigate the effects of NB-UVB on the expression of Gadd45α as well as cell apoptosis and cycle of human HaCaT keratinocytes.Methods Cultured HaCaT cells were exposed to various doses (100,200,400 mJ/cm2)of NB-UVB followed by an additional culture of 6,12 and 24 hours,respectively.Reverse transcription PCR and Western blot were performed to detect the mRNA and protein expression of Gadd45α respectively in HaCaT cells,cell counting kit 8 (CCK8)to measure the proliferation of cells,and flow cytometry to determine the cell cycle distribution of HaCaT cells before and after the exposure to NB-UVB.Results Gadd45α was expressed in HaCaT cells.After exposure to NB-UVB of the three doses,the mRNA and protein levels of Gadd45α increased at 6 hours and 12 hours,but declined at 24 hours,and significant changes were observed in HaCaT cells at the three time points after exposure to NB-UVB of the three doses (all P<0.05).The Gadd45α/β-actin mRNA ratio was 1.4360±0.6551.1.8633±0.0979,1.9266±0.1724 in HaCaT cells 12 hours after irradiation to NB-UVB of 100,200 and 400 mJ/cm2,respectively,significantly higher than that in unirradiated cells(0.6000±0.1276,all P<0.05).Also,increased Gadd45α/β-actin protein ratio was noted in HaCaT cells 12 hours after irradiation to NB-UVB of 100,200 and 400 mJ/cm2 compared with unirradiated cells (0.0773±0.0005,0.1936±0.0015,0.2373±0.0015 vs.0.0290±0.0010,all P<0.05).NB-UVB inhibited the proliferation of HaCaT cells in a time-and dose-dependent manner.Flow cytometry showed that irradiated HaCaT cells were blocked in G2 phase of the cell cycle.and the percentage of HaCaT cells in G2 phase was 13.53%±1.03%,17.77%±2.25%,30.03%±4.29%afler exposure to NB-UVB of 100,200 and 400 mJ/cm2,respectively,compared to 9.24%±0.97%in unirradiated cells (all P<0.05).Conclusions The expression of Gadd45α is increased in HaCaT cells after exposure to NB-UVB,and Gadd45α may be involved in the NB-UVB-induced suprression of cell proliferation of and cell cycle arrest in HaCaT cells.