WANG Lu,ZHANG Zheng,GAO Li,et al.Effects of Overexpression of YAP on Hydrogen Peroxide Induced Oxidative Damage in Human Dermal Fibroblasts[J].The Chinese Journal of Dermatovenereology,2019,(05):501-506.[doi:10.13735/j.cjdv.1001-7089.201808148]





Effects of Overexpression of YAP on Hydrogen Peroxide Induced Oxidative Damage in Human Dermal Fibroblasts
王露1张政2 高丽3 陈华全3周发忠3
1.四川省妇幼保健院皮肤科,四川 成都 610031;2.成都市第三人民医院,四川 成都 610031;3.德阳市人民医院皮肤科,四川 德阳 618000
WANG Lu1ZHANG Zheng2 GAO Li3CHEN Huaquan3 ZHOU Fazhong3
(1.Department of Dermatology, Sichuan Provincial Maternal and Child Care Service Centre, Chengdu 610031, China;2.Department of Neurology, the Third People′s Hospital of Chengdu, Chengdu 610031, China;3.Department of Dermatology, Deyang People′s Hospital, Deyang 618000, China)
Skin fibroblastsApoptosisOxidative damageYAP
R 751
目的研究过表达Yes相关蛋白(YAP)对过氧化氢(H2O2)诱导皮肤成纤维细胞氧化损伤的影响。方法皮肤成纤维细胞中转染pcDNA3.1-YAP质粒、对照pcDNA3.1质粒,经过H2O2处理后记为pcDNA3.1-YAP+H2O2、pcDNA3.1+H2O2,同时设置不转染的皮肤成纤维细胞经H2O2处理后记为H2O2,以不做任何处理的皮肤成纤维细胞作为对照。Real-time PCR和Western blot方法检测细胞中YAP表达变化,MTT检测细胞增殖,2,4-二硝基苯肼显色法检测乳酸脱氢酶(LDH)漏出率,流式细胞术检测细胞凋亡,Western blot检测细胞中活化型Caspase-3(Cleaved Caspase-3)蛋白水平,钼酸铵比色法检测过氧化氢酶(CAT)活性,硫代巴比妥酸法检测细胞中丙二醛(MDA)含量,黄嘌呤氧化酶法检测超氧化物歧化酶 (SOD)活性,二硫对二硝基苯甲酸比色法检测谷胱甘肽过氧化物酶(GSH-PX)活性,羟脯氨酸法检测胶原合成水平。结果pcDNA3.1-YAP+H2O2细胞中YAP水平高于pcDNA3.1+H2O2和对照(P<0.05)。H2O2细胞增殖能力降低,LDH漏出率升高,细胞凋亡率升高,细胞中Cleaved Caspase-3蛋白水平升高,CAT活性、SOD活性、GSH-PX活性降低,细胞中MDA含量升高,胶原含量降低,与对照比较,差异有统计学意义(P<0.05)。pcDNA3.1-YAP+H2O2细胞增殖能力升高,LDH漏出率降低,细胞凋亡率降低,细胞中Cleaved Caspase-3蛋白水平降低,CAT活性、SOD活性、GSH-PX活性升高,细胞中MDA含量降低,胶原含量升高,与pcDNA3.1+H2O2和H2O2比较,差异有统计学意义(P<0.05)。结论过表达YAP可以减轻H2O2诱导的皮肤成纤维细胞氧化损伤,减少细胞凋亡。
Objective To study the effect of overexpression of YAP on oxidative damage induced by hydrogen peroxide in human dermal fibroblasts.Methods pcDNA3.1-YAP plasmid and pcDNA3.1 plasmid were transfected into skin fibroblasts.Treatments of fibroblasts included pcDNA3.1-YAP+H2O2, pcDNA3.1+H2O2 as well as H2O2 alone.Fibroblasts with no treatment served as control.Real-time PCR and Western blot were used to assess the expression levels of YAP, while MTT was used to assess cell proliferation.The leakage rate of LDH and cell apoptosis were measured using 2,4- two nitrophenyl hydrazine colorimetric assay and flow cytometry, respectively.Western blot was used to measure the levels of cleaved Caspase-3.CAT and SOD activities were measured using ammonium molybdate colorimetric assay and xanthine oxidase assay, respectively.MDA content was determined using thiobarbituric acid method.Dithiobis 2-nitrobenzoic acid was used to measure GSH-PX activity.Hydroxyproline method was used to assess collagen synthesis.ResultsThe level of YAP in the cells treated with pcDNA3.1-YAP+H2O2 was higher than that in pcDNA3.1+H2O2 and control (P<0.05).In comparison to untreated controls, treatment with H2O2 decreased cell proliferation, activities of CAT, SOD and GSH-PX, and collagen content, while increasing the leakage rate of LDH, apoptosis, the level of cleaved Caspase-3 protein, MDA content (P<0.05 versus controls for all).However, overexpression of YAP overcame the alterations in these biomarkers above induced by H2O2 (P<0.05 versus both pcDNA3.1+H2O2 and H2O2 for all).ConclusionOverexpression of YAP can alleviate H2O2 induced oxidative damage in dermal fibroblasts.


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[作者单位]1.四川省妇幼保健院皮肤科,四川 成都 610031;2.成都市第三人民医院,四川 成都 610031;3.德阳市人民医院皮肤科,四川 德阳 618000
更新日期/Last Update: 2019-04-15