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上调miR-21对长波紫外线诱导光损伤模型人皮肤成纤维细胞的生物学作用

Biological Effects of Up-Regulated miR-21 on Human Skin Fibroblasts of Light Damage Model Induced by Ultraviolet A
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摘要 目的探究上调微小RNA-21(miR-21)对长波紫外线(UVA)诱导光损伤模型人皮肤成纤维细胞(human skin fibroblasts,HSF)生物活性、氧化损伤及磷酸酶和张力蛋白同源物(PTEN)/磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路的影响。方法将HSF培养至对数生长期,分为空白对照组、UVA组(使用UVA照射细胞7 d)、UVA+NC组[使用UVA照射细胞7 d,加入miR-21阴性对照(NC)慢病毒载体]、UVA+miR-21模拟物(mimics)组(使用UVA照射细胞7 d,加入miR-21 mimics慢病毒载体)、UVA+miR-21 mimics+PI3K抑制剂组(使用UVA照射细胞7 d,加入miR-21 mimics慢病毒载体和50μmol/L PI3K抑制剂LY294002)、UVA+miR-21 mimics+PI3K激活剂组(使用UVA照射细胞7 d,加入miR-21 mimics慢病毒载体和50μmol/L PI3K激活剂740Y-P)。各组均培养24 h。荧光定量PCR法检测各组HSF miR-21表达水平;细胞计数试剂盒-8(CCK-8)、流式细胞仪分别检测HSF活力、凋亡;β-半乳糖苷酶染色法观察HSF衰老情况;酶联免疫吸附法检测HSF中超氧化物歧化酶(SOD)、丙二醛(MDA)含量;蛋白印迹法检测HSF中PTEN/PI3K/AKT通路相关蛋白表达。结果与空白对照组相比,UVA组、UVA+NC组HSF中miR-21表达水平、活力、SOD含量、PI3K、磷酸化AKT(p-AKT)蛋白表达水平显著降低(P<0.05),凋亡率、衰老细胞比例、MDA含量、PTEN蛋白表达水平显著升高(P<0.05);与UVA组和UVA+NC组相比,UVA+miR-21 mimics组HSF中miR-21表达水平、活力、SOD含量、PI3K、p-AKT蛋白表达水平显著升高(P<0.05),凋亡率、衰老细胞比例、MDA含量、PTEN蛋白表达水平显著降低(P<0.05);PI3K抑制剂LY294002可逆转miR-21 mimics对UVA诱导的HSF损伤的保护作用(P<0.05);PI3K激活剂740Y-P可增强miR-21 mimics对UVA诱导的HSF损伤的保护效果(P<0.05)。结论上调miR-21可增强UVA诱导光损伤模型HSF活力,抑制其凋亡、衰老和氧化损伤,可能与调控PTEN/PI3K/AKT信号通路表达有关。 Objective To investigate the effects of up-regulated microRNA-21(miR-21)on biological activity,oxidative damage and phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)signaling pathway of light damage model human skin fibroblasts(HSF)induced by ultraviolet A(UVA).Methods HSF were cultured to logarithmic growth stage and divided into blank control group,UVA group(the cells were irradiated with UVA for 7 days),UVA+NC group[the cells were irradiated with UVA for 7 days and the lentivirus vector of miR-21 negative control(NC)was added],UVA+miR-21 mimics group(the cells were irradiated with UVA for 7 days and the miR-21 mimics lentivirus vector was added),UVA+miR-21 mimics+PI3K inhibitor group(the cells were irradiated with UVA for 7 days,the miR-21 mimics lentivirus vector and 50μmol/L PI3K inhibitor LY294002 were added),UVA+miR-21 mimics+PI3K activator group(the cells were irradiated with UVA for 7 days,the miR-21 mimics lentiviral vector and 50μmol/L PI3K activator 740Y-P were added).All groups were cultured for 24 h.Fluorescence quantitative PCR was used to detect the miR-21 expression level of HSF in each group;cell count kit-8(CCK-8)and flow cytometry were used to detect the viability and apoptosis of HSF;the senescence of HSF was observed byβ-galactosidase staining;the contents of superoxide dismutase(SOD)and malondialdehyde(MDA)in HSF were detected by enzyme-linked immunosorbent assay;Western blot was used to detect the expression of PTEN/PI3K/AKT pathway related proteins in HSF.Results Compared with blank control group,the expression level of miR-21,activity,SOD content,PI3K,phosphorylated AKT(p-AKT)protein expression levels of HSF in UVA group and UVA+NC group were significantly decreased(P<0.05),the apoptosis rate,proportion of senescent cells,MDA content and PTEN protein expression level were significantly increased(P<0.05);compared with UVA group and UVA+NC group,the expression level of miR-21,activity,SOD content,PI3K,p-AKT protein e
作者 齐立攀 赵亚平 刘竞 闫军飞 庞利霞 QI Lipan;ZHAO Yaping;LIU Jing;YAN Junfei;PANG Lixia(Department of Plastic Surgery,People's Hospital Affiliated to Hebei Medical University,Shijiazhuang People's Hospital,Shijiazhuang 050030,China)
出处 《中国皮肤性病学杂志》 CAS CSCD 北大核心 2023年第6期631-638,共8页 The Chinese Journal of Dermatovenereology
基金 河北省医学科学研究课题计划项目(20200134)。
关键词 微小RNA-21 长波紫外线 人皮肤成纤维细胞 生物活性 氧化损伤 PTEN/PI3K/AKT通路 MicroRNA-21 Ultraviolet A Human skin fibroblasts Biological activity Oxidative damage PTEN/PI3K/AKT pathway
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