氧化应激所致的人皮肤成纤维细胞衰老中β-连环蛋白的表达研究

摘要/Abstract

摘要：

目的观察β-连环蛋白在氧化应激所致的人皮肤成纤维细胞衰老中的变化，探讨其在应激诱导的皮肤衰老中可能的作用。方法从儿童包皮分离培养原代成纤维细胞，H2O2处理第2 ～ 4代人皮肤成纤维细胞，显微镜观察其形态学改变，β-半乳糖苷酶细胞衰老试剂盒检测β-半乳糖苷酶活性，RT-PCR、Western印迹检测β连环蛋白mRNA和蛋白表达水平。结果 150 μmol/L H2O2处理2 h后可以诱导人皮肤成纤维细胞衰老。在应激诱导的衰老（SIPS）细胞中β-半乳糖苷酶阳性率达到（37.67 ± 1.53）%，而对照细胞中只有（2.97 ± 0.25）%，两组差异有统计学意义（P < 0.01）。RT-PCR结果显示，在对照细胞组β连环蛋白/GAPDH mRNA比值为0.59 ± 0.04，SIPS细胞组为0.29 ± 0.30，对照细胞组明显高于SIPS细胞组，两组差异有统计学意义（t = 10.06，P < 0.01）。Western印迹结果显示，对照细胞组β连环蛋白/GAPDH蛋白比值为0.62 ± 0.03，SIPS细胞组为0.31 ± 0.01，对照细胞组明显高于SIPS细胞组，两组差异有统计学意义（t = 14.97，P < 0.01）。结论 150 μmol/L H2O2处理2 h可以诱导人皮肤成纤维细胞发生衰老改变，SIPS细胞中β连环蛋白表达水平明显降低，初步推测β连环蛋白可能是调控皮肤衰老的重要靶基因。

关键词: 人皮肤成纤维细胞

Abstract:

Objective To observe the changes of β-catenin expression in human skin fibroblasts （HSFs） after induced by oxidative stress, and to explore its possible roles in oxidative stress-induced premature senescence （SIPS） of HSFs. Methods Fibroblasts were isolated from the foreskin of a child and subjected to a primary culture. The fibroblasts of second to fourth passage were treated with various concentrations of H2O2 for 2 hours to establish an optimized model of stress-induced premature senescence. β-galactosidase assay kit was used to detect the activity of β-galactosidase in H2O2-induced HSFs, RT-PCR and Western blot to measure the mRNA and protein expressions of β-catenin in control and senescent HSFs. Results Premature senescence of HSFs could be induced by the treatment with H2O2 of 150 μmol/L for 2 hours. The proportion of β-galactosidase-positive cells was （2.97 ± 0.25）% in control HSFs and （37.67 ± 1.53）% in senescent HSFs （P < 0.01）. A significant increase was observed in the β-catenin/GAPDH protein ratio and β-catenin/GAPDH mRNA ratio in control HSFs compared with the senescent HSFs （0.62 ± 0.03 vs. 0.31 ± 0.01, t = 14.97, P < 0.01; 0.59 ± 0.04 vs. 0.29 ± 0.30, t = 10.06, P < 0.01）. Conclusions The two-hour treatment with H2O2 of 150 μmol/L could induce the premature senescence of HSFs, and there is a notable decrease in the expression of β-catenin in prematurely senescent HSFs induced by oxidative stress, implying that β-catenin is an important target gene for the regulation of skin aging.

Key words: human skin fibroblasts

田黎明谢红付李吉杨婷王玮蓁胡耀华. 氧化应激所致的人皮肤成纤维细胞衰老中β-连环蛋白的表达研究[J]. 中华皮肤科杂志, 2011, 44(4): 259-262.

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