紫外线诱导人角质形成细胞衰老的作用与端粒酶表达无关

Telomerase expression is not involved in aging process of human keratinocytes induced by UVB irradiation

目的:探讨人永生化角质形成细胞(HaCaT)和人皮肤原代角质形成细胞(HEKa)接受紫外线照射后的衰老机制及其凋亡、坏死和细胞周期的改变。方法:中波紫外线(UVB)小剂量、多次照射HaCaT细胞和HEKa细胞,以细胞衰老β-半乳糖苷酶(SA-β-Gal)化学染色法观察细胞衰老状态,流式细胞术检测细胞凋亡、坏死和细胞周期阻滞情况;ELISA法检测细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平;Western blot检测redox蛋白p66Shc的表达;RT-PCR法检测细胞内人端粒酶催化亚单位(human telomerase reverse transcriptase,hTERT)基因在UVB照射前后的表达情况。结果:UVB照射后,SA-β-Gal细胞化学染色结果显示HaCaT细胞和HEKa细胞均呈现强阳性染色。HaCaT细胞的凋亡率从(1.81±0.25)%增至(4.43±0.28)%,坏死率从(0.05±0.01)%上升至(0.10±0.03)%,但细胞周期未见明显阻滞;HEKa细胞的凋亡率从(0.65±0.05)%增至(59.53±2.35)%,坏死率达(3.89±0.24)%,较对照组显著增加(P<0.05),且细胞周期大部分阻滞于G0/G1期。HaCaT细胞和HEKa细胞的细胞内SOD活性经UVB照射后显著下降(P<0.05),而MDA水平则明显增高。p66Shc蛋白在UVB照射后表达增强。HaCaT细胞在UVB照射前后均高表达hTERT基因,而HEKa细胞不表达hTERT基因。结论:HaCaT细胞和HEKa细胞均可被UVB诱导进入"应激性早衰"状态,此衰老与细胞内氧化应激水平密切相关,而与端粒酶表达无关;但HaCaT细胞的凋亡率、坏死率及细胞周期阻滞情况较HEKa细胞好,这可能与HaCaT细胞高表达端粒酶水平,从而对细胞损伤修复能力及分化调节能力较强有关。

Objective： To investigate the mechanism involved in aging process of immortalized human keratinoeyte （HaCaT） and primary human epidermis keratinocyte of adults （HEKa） irradiated by ultraviolet B（UVB）. Methods： HEKa and HaCaT were repeatedly exposed to UVB at a subcytotoxic level. SA-β-Gal staining was performed to evaluate the senescence state; flow cytometry was applied to detect the changes of apoptosis, necrosis and cell cycle. Intracellular levels of superoxide dismutase （SOD） and malondialdehyde （MDA） were measured by ELISA method. Western blot was performed to detect the expression pattern of redox protein p66Shc and RT-PCR was performed to determine the mRNA level of human telomerase reverse transcriptase （hTERT）. Results： Strong positive SA-β-Gal staining was observed in both HEKa cell and HaCaT cells after UVB irradiation. Apoptosis rate increased from （1.81 ± 0. 25）% to （4.43 ± 0.28）% and necrosis rate increased from （0.05±0.01） %to （0.10±0.03）% in HaCaT cell,but no marked arrest of cell cycle was observed during UVB irradiation. As a contrast,apoptosis rate of in HEKa cells significantly increased from（0.65±0.05）% to （ 59. 53 ± 2. 35 ） % , and the necrosis rate in HEKa cells also reached （3.89±0.24）% （P〈0. 05）. Growth arrest in G0/G1 phase was also found in HEKa cells. In both cell lines,intracellular level of SOD decreased and MDA increased remarkably after UVB exposure,and an increased expression of p66Shc protein was also observed. High level of hTERT mRNA was detected in HaCaT cells and UVB exposure had little effect on its expression. Conclusion： The stress-induced premature senescence （SIPS） in HaCaT and HEKa cell lines by UVB irradiation might be closely associated with increased intracellular levels of oxidative stress,not related to the telomerase expression.