CaM对小鼠胎儿成纤维细胞Hsp70表达的影响

Effect of CaM on Expression of Inducible Hsp70 in MEF

【目的】证实小鼠胎儿成纤维细胞中钙调蛋白(calmodulin,CaM)基因参与热应激诱导型热休克蛋白70(Hsp70)的基因表达并明确其作用条件。【方法】取12.5 d孕鼠胚胎制备成纤维细胞(MEF),上述细胞随机分为对照组和热应激组:对照组在37℃条件下培养,热应激组包括Ⅰ组(39℃、0.5 h),Ⅱ组(39℃、1 h),Ⅲ组(39℃、1.5 h),Ⅳ组(41℃、0.5 h),Ⅴ组(41℃、1 h),Ⅵ组(41℃、1.5 h);每组3个重复。将选择的热应激组和对照组细胞分别加入不同浓度(25、50及100 mmol.L-1)钙调蛋白拮抗剂W7,运用RT-PCR检测Hsp70、CaM mRNA表达量。【结果】39℃和41℃热应激组Hsp70 mRNA表达量显著高于常温对照组(P<0.05);39℃应激1h的Hsp70 mRNA表达量极显著高于其它各组(P<0.01);39℃应激0.5 h和1 h CaM mRNA表达量极显著高于其它各组(P<0.01);41℃热应激组的CaM mRNA表达量与常温对照组比较差异不显著(P>0.05)。在培养液中加100 mmol.L-1W7、分别于37℃和39℃处理1 h后,其成纤维细胞的Hsp70 mRNA表达量极显著低于相应对照组(P<0.01)。【结论】中度热应激条件下,Hsp70和CaM基因表达量呈正相关,且39℃应激1 h可以显著诱导Hsp70和CaM基因表达;CaM通过某种途径参与了Hsp70基因表达。

【Objective】The objectives of this study were to reveal whether or not the calmodulin（CaM） gene participates in HSP70 gene expression in the mouse fetal fibroblasts and to definite their participating conditions.【Method】 The 12.5-day mouse embryo of pregnancy was taken to culture its fibroblasts（MEF）.The fibroblasts were divided into control group and heat shock group randomly.The heat shock group was further divided into groups of Ⅰ（39℃ for 0.5 h）,Ⅱ（39℃ for 1 h）,Ⅲ（39℃ for 1.5 h）,Ⅳ（41℃ for 0.5 h）,Ⅴ（41℃ for 1 h） and Ⅵ（41℃ for 1.5 h）,respectively,while the control group was treated at 37℃.Three replicates were performed for each group.The optimum heat stress group was then selected.Calmodulin antagonist W7 was applied to both the selected and control group with different concentrations（25,50,and 100 mmol·L-1） in parallel.Both expressions of Hsp70 and CaM mRNA were detected by using the method of RT-PCR.【Result】 The expressions of Hsp70 mRNA in both 39℃ and 41℃ groups were significantly higher than that of the control group（P〈0.05）.However,the expression of Hsp70 mRNA in the group treated at 39℃ for 1h was significantly higher compared with other groups（P〈0.01）.The expressions of CaM mRNA in the groups treated both at 39℃ for 0.5 h and 39℃ for 1 h were significantly higher than that in other groups（P〈0.01）.There was no difference in CaM mRNA expression in the group treated at 41℃ compared with the control group（P〈0.05）.The expressions of Hsp70 mRNA in the groups of 37℃ and 39℃ for 1 h treated with 100 mmol·L-1 W7 were significantly lower than that in corresponding control group（P〈0.01）.【Conclusion】 There is a positive correlation between Hsp70 and CaM gene expression under moderate heat shock and the treatment at 39℃ for 1 h induced both Hsp70 and CaM gene expressions.The CaM gene participates in HSP70 gene expression.