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目的研究中波紫外线(UVB)辐射对表皮角质形成细胞核因子 kB(NF-kB)和 P53信号通路的影响以及 NF-kB 和 P53信号通路的交互作用。方法正常人表皮角质形成细胞(NHEK)(含野生型 p53)和永生化人角质形成细胞株 HaCaT(含突变型 p53)各分2组于37℃、5%CO_2环境培养。当细胞融合达85%时进行 UVB(60 mJ/cm~2)辐射,其中1组于辐射前1 h 加入终浓度为5μmol/L 具有抗 NF-kB 活化作用的 BAY11-7082。分别提取辐射前后 NHEK 和 HaCaT 细胞蛋白和核蛋白,应用 Western 印迹检测 NF-kB、P53和 P21蛋白的表达水平,应用电泳迁移变更分析(EMSA)检测NF-kB 的转录情况。结果 UVB 辐射可激活 NHEK 和 HaCaT 的 NF-kB、P53和 P21的蛋白表达和NF-kB 的转录活性。BAY11-7082对 NHEK 和 HaCaT 的 NF-kB 的转录活性均具有显著的抑制作用。作为 NF-kB 的抑制剂,BAY11-7082还显著抑制了 NHEK 的 P53和 P21的蛋白表达(P53:0.08±0.07vs 0.78±0.32,P<0.01;P21:0.65±0.22 vs 1.58±0.77,P<0.05),但不影响 HaCaT 的 P53和 P21的蛋白表达。结论 UVB 辐射激活表皮角质形成细胞的 NF-kB 和 P53信号通路存在交互作用,这种交互作用与 P53功能状况相关。
Objective To investigate the effects of ultraviolet (UVB) radiation on the expression of NF-κB and P53 in epidermal keratinocytes and the interaction between NF-κB and P53 signaling. Methods Normal human epidermal keratinocytes (NHEK) (including wild-type p53) and immortalized human keratinocyte cell line HaCaT (mutant p53-containing) were divided into two groups and cultured in the environment of 37 ℃ and 5% CO 2. UVB irradiation (60 mJ / cm ~ 2) was performed when the cells reached 85% confluence. One group was exposed to BAY11-7082 at a final concentration of 5 μmol / L for 1 h before irradiation. The protein and nuclear protein of NHEK and HaCaT were extracted before and after irradiation. The expression of NF-kB, P53 and P21 protein were detected by Western blot. The transcription of NF-kB was detected by electrophoretic mobility shift assay (EMSA). Results UVB radiation activated NF-kB, P53 and P21 protein expression and NF-kB transcriptional activity of NHEK and HaCaT. BAY11-7082 has a significant inhibitory effect on the NF-kB transcriptional activity of NHEK and HaCaT. As an inhibitor of NF-kB, BAY11-7082 also significantly inhibited the protein expression of P53 and P21 of NHEK (P53: 0.08 ± 0.07 vs 0.78 ± 0.32, P <0.01; P21: 0.65 ± 0.22 vs 1.58 ± 0.77, P < 0.05), but did not affect the protein expression of P53 and P21 in HaCaT. Conclusion There is interaction between NF-kB and P53 signaling pathway activated by UVB radiation in epidermal keratinocytes. This interaction is related to the functional status of P53.