中文摘要：

目的 初步研究14-3-3σ在中波紫外线（UVB）照射诱导的HaCaT细胞增殖和周期阻滞中的作用。方法 不同剂量UVB（0、10、20、30、40、50、60和80 mJ/cm2）照射HaCaT细胞和14-3-3σ稳定干扰细胞系（HaCaTKD14-3-3σ,以下简称HaCaTKD）。照后48 h,结晶紫染色法观察细胞生长密度。30 mJ/cm2UVB照射后,CCK-8法检测HaCaT和HaCaTKD细胞的增殖变化,流式细胞术分析细胞G2/M期细胞比例的变化,Western blot检测UVB照射后两种细胞的14-3-3σ、Cdc2、Cdc25c和Cyclin B1蛋白表达水平。结果 UVB照后48 h,两种细胞存活率均呈剂量依赖性下降,且HaCaTKD在10 mJ/cm2即可出现明显下降（t=8.83~49.63,P〈0.05）。HaCaTKD细胞的增殖能力显著低于HaCaT细胞（F=32.89,P〈0.05）;30 mJ/cm2的UVB照射后,HaCaT细胞在24 h后恢复增殖能力,但HaCaTKD细胞在72 h仍未见增殖变化。HaCaTKD细胞在各观察点G2/M期细胞比例均未改变（P〉0.05）;30 mJ/cm2UVB照射后,HaCaT细胞在6~18 h发生G2/M期阻滞（t=7.41、9.22、9.16,P〈0.05）,HaCaTKD细胞G2/M期细胞比例未发生改变（P〉0.05）。UVB照后6、12、18、24 h和照前相比,HaCaT细胞中14-3-3σ表达量上调（t=5.42~9.57,P〈0.05）;HaCaT和HaCaTKD两种细胞中的Cdc25c和Cyclin B1的表达水平在照后均下调（t=3.95~11.21,P〈0.05）;Cdc2在HaCaT细胞中下调（t=4.93~5.37,P〈0.05）,在HaCaTKD细胞中无变化（P〉0.05）。结论 UVB照射或不照射,14-3-3σ的表达情况均能影响HaCaT细胞的增殖变化和细胞周期,UVB照射能诱导HaCaT细胞发生G2/M期阻滞,这种阻滞可能是由14-3-3σ的表达所介导,进而引起周期相关蛋白Cdc2、Cdc25c和Cyclin B1的表达改变而引发的。

英文摘要：

Objective To explore the role of 14-3-3σ in cell cycle arrest, proliferation inhibition of HaCaT cells after UVB exposure. Methods Crystal violet assay was used to determine the viable density of HaCaT, HaCaTKD cells after being irradiated with UVB of different doses（10,20,30,40,50,60 and 80 mJ/cm2）for 48 h. After HaCaT and HaCaTKD being treated with 30 mJ/cm2 irradiation, cell growth and cell cycle distribution were detected by CCK-8 assay and PI staining combined with flow cytometry, respectively. Western blot was used to evaluate the protein expression of 14-3-3σ, Cdc2, Cdc25c and Cyclin B1. Results After 48 h, the survival rate of both HaCaT and HaCaTKD decreased in a dose-dependent manner. Especially, HaCaTKD cells had drastically low proliferation rate compared with normal HaCaT at 10 mJ/cm2 （t=8.83-49.63,P〈0.05）. The proliferation rate of HaCaTKD cells was significantly lower than that of HaCaT cells（F=32.89,P〈0.05）. After treatment with 30 mJ/cm2 UV irradiation, the ability of proliferation in normal HaCaT cells was recovered after 24 h while there was no proliferation in HaCaTKD cells within 72 h after the same treatment. The distribution of cell cycle has little change in HaCaTKD（P〉0.05）. UVB treatment led to cell cycle arrest from 6 to 18 h in HaCaT cells（t=7.41, 9.22, 9.16, P〈0.05）while no cell cycle arrest could be observed in the HaCaTKD cell. Western blot detection indicated that the expression of 14-3-3σ in HaCaT cells was upregulated（t=5.42-9.57,P〈0.05）while the Cdc25c and Cyclin B1 proteins were downregulated in both HaCaT and HaCaTKD cells（t=3.95-11.21,P〈0.05）. Specifically, Cdc2 protein decreased in HaCaT cells（t=4.93-5.37,P〈0.05）but there was no change in HaCaTKD cells. Conclusions 14-3-3σ protein affects the proliferation and cell cycle of HaCaT cells after UVB irradiation. 14-3-3σ co-activates the expression of Cdc2, Cdc25c and Cyclin B1 to mediate UVB-induced G2/M arrest in HaCaT cells.