紫外线诱导角质形成细胞凋亡和表没食子儿茶酚没食子酸酯保护机理的探讨

Study on the Mechanism of UVB Irradiation and(-)-epigallocatechin-3-gallate Protection on Apoptosis of Cultured Human Keratinocytes

目的探讨不同剂量的中波紫外线(UVB)辐射对体外培养的人角质形成细胞(HaCaT细胞株)凋亡和死亡的影响,探讨表没食子儿茶酚没食子酸酯(EGCG)对此影响的保护作用,以及与凋亡相关的bcl-2和Fas基因之间的关系。方法流式细胞仪检测不同剂量UVB辐照及EGCG处理后角质形成细胞凋亡和死亡数量以及bcl-2蛋白水平的变化,RT-PCR检测FasmRNA的表达水平。结果UVB辐照组凋亡细胞数和死亡细胞数显著高于对照组(P<0.01),bcl-2和FasmRNA量与对照组差异也有显著性(P<0.01)。UVB辐照126mJ/cm2组凋亡细胞数低于辐照42mJ/cm2组(t=2.39,P<0.05),但死亡细胞比例显著增加(t=4.82,P<0.01),两组bcl-2和FasmRNA表达量差异均无显著性(t=1.30,P>0.25;t=0.32,P>0.25)。UVB辐照42mJ/cm2立即加入EGCG组较辐照42mJ/cm2未加EGCG组凋亡和死亡细胞数降低(t=7.64,P<0.01;t=3.49,P<0.05),bcl-2增加(t=3.62,P<0.05),FasmRNA表达量减少(t=6.83,P<0.01)。结论小剂量UVB辐照可以诱导体外培养的角质形成细胞凋亡,大剂量UVB辐照则导致细胞死亡,EGCG通过与凋亡相关的bcl-2和Fas减少紫外线诱导的角质形成细胞凋亡。

Objective To investigate the effects of UVB irradiation and(-)-epigallocatechin-3-gallate(EGCG)protection on apoptosis and death of cultured human keratinocytes(HaCaT cell line),and its association with bcl-2and Fas gene.Methods Flow cytometry was applied to detect the number of apop-totic and dead keratinocytes and the level of bcl-2protein.Fas mRNA was measured by RT-PCR method.Results The numbers of apoptotic and dead cells were significantly higher in all of the UVB irradiation groups than those in normal controls(P <0.01).Compared with42mJ/cm 2 UVB groups,the number of apoptotic cells was much lower(t=2.39,P<0.05)and the number of dead cells was much higher(t=4.82,P<0.01)in126mJ/cm 2 UVB group.However,there were no significant difference of bcl-2(t=1.30,P>0.25)and Fas mRNA(t=0.32,P>0.25)levels in the two groups.In the group treated with42mJ/cm 2 UVB irradiation followed by the addition of EGCG,the numbers of apoptotic and dead cells and Fas mRNA were decreased,but bcl-2protein was increased.Conclusions Low dosage of UVB irradiation could induce apoptosis of keratinocytes.High dosage of UVB irradiation might result in cell death.EGCG could reduce UVB-induced apoptosis of keratinocytes by increasing bcl-2protein and decreasing Fas mRNA.