摘要
目的了解EGF通过过氧化物酶体增殖物激活受体β（PPARβ调控HaCaT细胞凋亡的作用。方法将培养的HaCaT细胞根据培养液中所加物质不同分为对照组（常规培养）；TNF-α组（加入10ng／mL TNF-α）；EGF组（加入20ng／mL EGF）；EGF＋TNF-α组（用20ng／mL EGF预处理后4h，再予以10ng／mL TNF—α处理60min）。采用凝胶电泳迁移率改变分析法（EMSA）及荧光素酶基因分析法检测各组细胞中PPARβ的结合活性及转录活性；将细胞转染错义寡核苷酸（scrODN）及反义寡核苷酸（asODN）后，采用蛋白质印迹法检测PPARβ蛋白的表达；应用流式细胞术检测半胱氨酸天冬氨酸蛋白酶3（caspase-3）活性及细胞凋产率。结果EMSA及荧光素酶基闰分析显示，各实验组PPARβ的DNA结合活性及转录性增强；蛋白质印迹法显示，与转染scrODN的各组细胞比较，转染asODN的各组PPARβ蛋白表达明显被抑制。转染asODN的TNF-α组及EGF＋TNF-α组caspase-3活性明显高于转染scrODN的TNF-α组及EGF＋TNF-α组（P〈0．01）。转染scrODN的对照组、EGF组、TNF-α组和EGF＋TNF-α组的细胞凋亡率分别为（7．31±0．45）％、（7．43±0．21）％、（39．78±0．65）％、（28．34±0．54）％，转染asODN的对照组、EGF组、TNF-α组、EGF＋TNF-α组的细胞凋亡率分别为（8．22±0．51）％、（7．83±0．67）％、（46．78±0．48）％、（44．69±0。83）％，其中转染asODN的TNF-α组、EGF＋TNF—α组的细胞凋亡率明显高于转染scrODN的TNF-α组、EGF＋TNF-α组（P〈0．01）。结论EGF通过PPARβ依赖的方式抑制炎性因子TNF—α，导致HaCaT细胞凋亡。
Objective To explore the role of EGF in regulating HaCaT apoptosis through peroxisome proliferator-aetivated receptor 13 （PPARβ）. Methods Cultured HaCaT cells were divided into different groups with different additives in culture medium as control （normal culture） , TNF-α （with addition of 10 ng/mL TNF-α） , EGF （ with addition of 20 ng/mL EGF） , EGF ＋ TNF-α （ cells were treated with 10 ng/mL TNF-α for 60 mins after the exposure to 20 ng/mL EGF for 4 hs） groups. Conjugation activity and transcription activity of PPARβ of HaCaT cells in each group were detected by electrophoretic mobility shift assay （EMSA） and luciferase gene analysis （LGA）. Protein expression of PPARβ of HaCaT cells after transfected by missense oligonucleotide （scrODN） and antisence oligonucleotide （asODN） was determined by Western blot. Caspase-3 activity and apoptosis rate were detected by flow cytometry. Results Conjugation and transcription activity of PPARβ DNA were enhanced as shown in EMSA and LGA. Compared with that of cells in groups transfected by scrODN, protein expression of PPARβ in cells of groups transfected by asODN was obviously inhibited as shown in Western blot. Caspase-3 activity of cells in TNF-α and EGF ＋TNF-α groups transfected by asODN was stronger than that of cells in TNF-α and EGF ＋ TNF-α groups transfected by scrODN （ P 〈 0.01 ）. Apoptosis rate of cells in control, EGF, TNF-α, and EGF ＋ TNF-α groups which were transfected by scrODN was （7.31 ±0.45）%, （7.43 ±0.21）%, （39.78 ±0.65）%, （28.34 ±0.54）% respectively, and that in those groups transfected by asODN was （8.22 ± 0.51 ）%,（7.83 ±0.67）% , （46.78±0.48）% , （44.69 ±0.83）%. Apoptosis rate of cells in TNF-α and EGF ＋ TNF-α groups transfeeted by asODN was respectively higher than that in TNF-α and EGF ＋ TNF-α groups transfeeted by serODN （ P 〈 0.01 ）. Conclusions EGF inhibits HaCaT KC apoptosis caused by TNF-α in a PPARβ-dependent manner.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2009年第4期294-297,共4页
Chinese Journal of Burns
