过氧化物酶体增殖物激活受体β在肿瘤坏死因子α介导HaCaT角质细胞凋亡中的作用  

The role of PPARβ in the apoptotic HaCaT keratinocytes induced by TNF-α

作　　者：梁鹏飞[1] 蒋碧梅[2] 张丕红[1] 杨兴华[1] 龙剑虹[1] 黄晓元[1] 

机构地区：[1]中南大学湘雅医院烧伤整形科,长沙410008 [2]中南大学基础医学院病理生理教研室

出　　处：《中华医学杂志》2007年第30期2144-2148,共5页National Medical Journal of China

摘　　要：目的探讨肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)导致 HaCaT 角质细胞凋亡中,TNF-α对过氧化物酶体增殖物激活受体β(peroxisome proliferator activated receptor-β,PPARB)表达时空和转录活性的影响。方法采用 Hoechst 33258染色后观察凋亡细胞的形态学改变,并计算凋亡细胞百分率,Caspase 活性定量检测试剂盒分析 Caspase-3的活性变化,RT-PCR 和 Western 印迹观察 TNF-α导致 PPARBmRNA 及蛋白的表达,应用凝胶迁移滞留实验及荧光素酶报道基因分析 TNF-α介导 PPARβ的 DNA 结合活性及转录活性的改变。结果 HaCaT 角质细胞经5、10、20 ng/ml TNF-α处理24 h 后,Hoechst 33258染色示随着 TNF-α剂量的增加,凋亡细胞增多,凋亡细胞核百分率分别为12%±3%、32%±4%、57%±5%;HaCaT 角质细胞经 TNF-α(10、20 ng/ml)处理不同时间后,Caspase-3的活性增强(P<0.01)。继而 Western 印迹显示,HaCaT 角质细胞经10 ng/ml TNF-α处理12、24 h后 PPARβ的蛋白表达显著增加,HaCaT 角质细胞经不同浓度的 TNF-α处理24 h 后 PPARβ蛋白表达水平亦增加;RT-PCR 检测示 TNF-α(10、20 ng/ml)处理3、6 h 后 PPARBmRNA 的表达增强,说明TNF-α可诱导 PPARB 的表达。进而凝胶迁移滞留实验及荧光素酶报道基因分析表明 TNF-α可以增强 PPARβ的 DNA 的结合活性和转录活性。结论在 TNF-α介导 HaCaT 角质细胞凋亡中,TNF-α可增强 PPARβ mRNA、蛋白水平的表达以及 DNA 结合活性和转录活性。Objective To explore the change of transcription activity and expression of PPARβ in the apoptotic HaCaT keratinocytes induced by . Methods HaCaT keratinocytes were exposed to different concentration TNF-α for 24 hours. Apoptotic morphological changes and percentage of apoptotic nuclei were assayed with Hoechst 33258 staining. Activities of Caspase-3 were analyzed with Caspase Colorimetric Assay Kit after HaCaT keratinocytes were exposed to TNF-α（ 10 and 20 ng/ml） for indicated durations. The expression of PPARβ in HaCaT keratinocytes treated with TNF-α was observed by Westernblot and RT-PCR. Electrophoretic mobility shift assays demonstrated a impermanency increase in PPARβ binding activity with DNA. Furthermore, luciferase assay system were employed to analyze PPARβ transcription activity. Results The apeptosis of HaCaT keratinocytes treated with different concentration TNF-α for 24 hours was increased by Hoechst 33258 stained, and fluorescent microscopy showed apeptotic cells with condensed chromatin. The nuclear apoptotic percentage were （ 12 ± 3 ） %, （ 32 ± 4 ） %, （ 57± 5）%, respectively, in HaCaT keratinocytes exposed to TNF-α （5, 10, 20 ng/ml） for 24 hours. The activation of Caspase-3 were enhanced in HaCaT keratinocytes treated with TNF-α （ 10 or 20 ng/ml） for indicated durations （ P 〈 0. 01 ） . The expression of PPARβ protein significantly increased in HaCaT keratinocytes treated with TNF-α （ 10 ng/ml） for 12 and 24 hours. After exposure to different concentration of TNF-α for 24 hours, Western-blot analysis demonstrated to augment the expression of PPARβ in HaCaT keratinocytes. RT-PCR testified the expression of PPARβ mRNA is markedly increased in HaCaT keratinocytes treated with TNF-α （ 10,20 ng/ml） for 3 hours and 6 hours. PPARβ- DNA binding was assessed by EMSA using a PPARβ response element （PPRE） and nuclear extracts prepared from HaCaTkeratinocytes treated for 30 minutes and 60 minutes with 10 ng/ml of TNF-α demerstrated TNF-α enhanc

关 键 词：细胞凋亡 表皮 过氧化物酶体类 肿瘤坏死因子 

分 类 号：R363[医药卫生—病理学]