热休克因子在过氧化氢所致小鼠胚胎成纤维细胞损伤中的作用

Effect of Heat Shock Factor 1 (HSF1) on Hydrogen Peroxide-Mediated Impairment of Mouse Embryonic Fibroblasts (MEFs)

【目的】探讨热休克因子1(heat shock factor 1,HSF1)对过氧化氢所致小鼠胚胎成纤维细胞(mouseembryonic fibroblasts,MEFs)损伤的影响。【方法】利用HSF1基因敲除(HSF1-/-)小鼠和转基因技术,通过稳定转染SV40大T抗原基因,建立缺失HSF1基因(HSF1-/-纯合子)和含有HSF1基因(HSF1+/+野生型)的永生化MEFs,并采用免疫印迹和基因组DNA PCR技术进行鉴定。向细胞培养基中加入终浓度为0.5 mmol/L的过氧化氢导致细胞损伤,采用甲苯胺兰染色和总蛋白合成能力分析方法来判定致细胞损伤程度(核仁的结构和功能改变)。【结果】PCR产物电泳显示在各种基因型MEFs中均含有SV40大T抗原基因,在HSF1-/-MEFs中仅含有替代基因,在HSF1+/+MEFs中仅含HSF1基因,在HSF1-/+MEFs中则含有替代基因和HSF1基因;免疫印迹显示热休克反应(heat shock response,HSR)不能诱导HSF1-/-MEFs中HSP70表达。甲苯胺兰染色和总蛋白合成能力分析显示0.5 mmol/L过氧化氢可引起HSF1+/+MEFs及HSF1-/-MEFs核仁分离及蛋白质合成抑制,与正常对照组相比,P<0.05;HSR能显著抑制过氧化氢所致HSF1+/+MEFs核仁分离及蛋白质合成抑制,与单纯过氧化氢损伤组相比,P<0.05,但HSR对过氧化氢所致HSF1-/-MEFs核仁分离及蛋白质合成抑制没有影响,与单纯过氧化氢损伤组相比,P>0.05。【结论】HSR以一种HSF1依赖方式显著抑制过氧化氢所致细胞核仁结构与功能损伤。

[Objective] To investigate the effect of heat shock factor 1 （HSF1） on hydrogen peroxide （H2 O2 ）- mediated impairment of mouse embryonic fibroblasts （MEFs）. [Methods] MEFs were isolated from HSF1 knock out （HSF1^-/- ） and wild type （HSF1^＋/＋ ） mice and immortalized by transfection of an eukaryotic expression vector containing SV40 large T antigen and screening with G418. Immunoblotting and polymerase chain response （PCR） for genomic DNA were performed to identify the immortal cell lines. Damage of cell was estimated by toluidine blue staining and total protein synthesis respectively. [Results] Eleetrophoresis of PCR products showed SV40 large T antigen in all MEFs with three genotypes, only the replacement gene in HSF1^-/- MEFs, only HSF1 gene in HSF1^＋/＋ MEF.s and both genes in HSF1^-/＋ MEFs. Immunoblotting revealed that heat shock response （HSR） could not induce the expression of heat shock protein 70 （HSPT0） in HSF1^-/- MEFs. Toluidine blue staining and total protein synthesis showed that 0. 5 mmol/L H2O2 could result in nucleolar segregation and inhibition of total protein synthesis in HSF1^＋/＋ MEFs and HSF1^-/- MEFs, compared with control group（ P 〈0.05）. HSR could suppress significantly H2O2-mediated nucleolar segregation and inhibition of total protein synthesis in HSF1^＋/＋ MEFs. However, HSR had no effect on H2O2 mediated nucleolar segregation and inhibition of total protein synthesis in HSF1^-/- MEFs. [Conclusion]HSR inhibits H2O2-mediated structural and functional impairment of nucleoli in MEFs in a HSF1-dependent manner.