UVB诱导人皮肤成纤维细胞中beta-catenin基因的变化研究

The study of beta-catenin change in human fibroblast induced by UVB irradiation

目的:观察中波紫外线(UVB)诱导人皮肤成纤维细胞中β-catenin及其下游基因c-myc的变化,探讨其在紫外线诱导光老化和肿瘤发生中的作用。方法:使用组织块法分离培养原代成纤维细胞,使用第2～8代人皮肤成纤维细胞进行紫外线处理,倒置荧光显微镜观察其形态学改变,β-gal半乳糖苷酶细胞衰老试剂盒染色衰老细胞、western blot检测衰老相关蛋白P16的表达,western blot检测紫外线处理人皮肤成纤维细胞各时相点β-catenin、c-myc蛋白表达水平,RT-PCR检测紫外线处理人皮肤成纤维细胞各时像点β-catenin、c-mycmRNA水平的表达变化。结果:40mJ/cm2UVB单次照射后可以诱导人皮肤成纤维细胞衰老,β-gal衰老染色阳性率达到约90%,衰老相关基因P16蛋白表达明显升高,β-catenin、c-myc蛋白表达水平和mRNA水平照射后较未照射组均明显降低。结论:紫外线UVB处理人皮肤成纤维细胞可以诱导其发生衰老改变,衰老细胞中β-catenin、c-myc表达水平明显降低,这些结果与皮肤黑色素细胞瘤相关基因改变的研究相近,为以后皮肤恶性肿瘤的研究提供了线索。

Objective To observe the change of beta-catenin and its follow gene c-myc in human fibroblast after induced by UVB,exploure their roles at UVB induced photoaging and cancer.Methods Isolation and culture human skin fibroblast by tissues methods,fibroblasts were irradiation at different dose UltravioletB,observation the cells＇morphology under the invert microscope.Senescence cells were stained by β-galactosidase kit,the level of P16 protein were measured by western blot.The protein and mRNA level of beta-catenin,c-myc by western blot at different time points after UVB irradiation.Results Fibroblast senescence can be induced by ultravioletB irradiation at 20、40 mJ/cm2,the stain positive ration arrived at about 90%.The protein level of P16 increased obviously following times.The protein and mRNA level of beta-catenin and c-myc decreased obviously after UVB irradiation compared to control.Conclusion The beta-catenin and c-myc gene decrease in Human skin fibroblasts after induced by ultravioletB irradiation.