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目的探讨14-3-3σ与TPD52L1在低剂量中波紫外线(UVB)诱导的HaCaT细胞凋亡、增殖和细胞周期中的作用。方法 1取对数生长期HaCaT细胞,予低剂量UVB(累计照射剂量为2.97×10~(-2)J/cm~2)照射后培养6~48h收获细胞,另设不予UVB照射(予假照射)的对照,采用流式细胞分析法检测细胞凋亡率;将HaCaT细胞分为对照组和UVB组,于相应照射处理后0~72 h收获细胞,采用噻唑蓝法检测细胞增殖能力。2取HaCaT细胞随机分为对照组(予假照射)和UVB组,于相应照射处理后0~24 h收获细胞,采用流式细胞术检测G2/M期细胞比例。3予低剂量UVB照射HaCaT细胞后培养3~24 h或3~30 h,收获细胞,另设予假照射的对照,采用实时荧光定量聚合酶链式反应检测细胞中14-3-3σ与TPD52L1的mRNA的相对表达水平,采用蛋白质印迹法检测细胞中14-3-3σ与TPD52L1的蛋白相对水平表达。结果 1 UVB照射后24 h,HaCaT细胞凋亡率上升至最高值(P<0.05),增殖能力下降至最低值(P<0.05)。2对照组HaCaT细胞G2/M期比例呈现在假照射后6~12 h维持在最高值,至假照射后18 h下降至最低值的周期性改变特征;UVB组HaCaT细胞G2/M期比例在照射后6、12和18 h均较对照组上升(P<0.05),并持续在最高值,发生较明显的G2/M期阻滞。3UVB照射后HaCaT细胞中14-3-3σ的mRNA和蛋白相对表达水平均呈现先上升后下降现象。其中,14-3-3σmRNA相对表达水平于照射后3 h上升至峰值(P<0.05),于照射后3~12 h维持峰值水平(P<0.05),其后逐渐下降,于照射后24 h恢复正常水平;14-3-3σ蛋白相对表达水平于照射后6 h开始上升(P<0.05),于照射后18 h上升至峰值(P<0.05),其后逐渐下降,但于照射后30 h仍未恢复正常水平。TPD52L1 mRNA相对表达水平则呈现先下降后上升现象,于照射后12 h下降至最低水平(P<0.05),其后逐渐升高,于照射后24 h上升至峰值(P<0.05);TPD52L1蛋白相对表达水平呈先上升后下降现象,于照射后24 h上升至峰值(P<0.05),于照射后30 h下降至正常水平(P<0.05)。结论 14-3-3σ和TPD52L1在低剂量UVB诱导HaCaT细胞发生凋亡、增殖和G2/M期阻滞中可能共同发挥重要作用。
Objective To investigate the role of 14-3-3σ and TPD52L1 in the apoptosis, proliferation and cell cycle of HaCaT cells induced by low dose UVB. Method 1 HaCaT cells were harvested at a low dose of UVB (cumulative radiation dose of 2.97 × 10 -2 J / cm 2) and cultured for 6-48 h. The cells were treated with UVB Sham irradiation). HaCaT cells were divided into control group and UVB group. Cells were harvested at 0 ~ 72 h after irradiation. Cell viability was measured by thiazolyl blue assay. 2 HaCaT cells were randomly divided into control group (pre-irradiation) and UVB group. Cells were harvested at 0-24 h after irradiation. The proportion of cells in G2 / M phase was detected by flow cytometry. 3 cells were irradiated with low dose of UVB and cultured for 3-24 h or 3-30 h. Cells were harvested and sham-irradiated controls were also established. Real-time fluorescent quantitative polymerase chain reaction was used to detect the expression of 14-3-3σ and TPD52L1 MRNA relative expression levels of 14-3-3σ and TPD52L1 proteins were detected by Western blot. Results 1 The apoptosis rate of HaCaT cells increased to the highest value (P <0.05) 24 h after UVB irradiation, and the proliferation ability decreased to the lowest (P <0.05). The percentage of G2 / M phase of HaCaT cells in control group was the highest at 6 ~ 12 h after sham irradiation, but decreased to the lowest at 18 h after sham irradiation. The percentage of G2 / M phase in HaCaT cells in UVB group was At 6, 12, and 18 h after irradiation, the cells were increased compared with the control group (P <0.05), and continued at the highest level, with obvious G2 / M arrest. After 3UVB irradiation, the expression level of 14-3-3σ mRNA and protein in HaCaT cells increased first and then decreased. Among them, the relative expression level of 14-3-3σmRNA peaked at 3 h (P <0.05) and peaked at 3 ~ 12 h after irradiation (P <0.05), and then decreased gradually. After 24 h (P <0.05). The relative expression level of 14-3-3σ protein increased at 6 h after irradiation (P <0.05), and reached its peak at 18 h after irradiation (P <0.05), and then decreased gradually. However, h has not returned to normal levels. The relative expression level of TPD52L1 mRNA decreased first and then decreased to the lowest level at 12 h after irradiation (P <0.05), then gradually increased and reached its peak at 24 h after irradiation (P <0.05). TPD52L1 protein The relative expression level increased first and then decreased, and reached the peak at 24 h after irradiation (P <0.05), and decreased to normal level at 30 h after irradiation (P <0.05). Conclusion 14-3-3σ and TPD52L1 may play an important role in apoptosis, proliferation and G2 / M arrest in HaCaT cells induced by low dose UVB.