热休克蛋白70过表达对过氧化氢诱导BRL细胞caspase 3表达的影响

Effect of overexpression of heat shock protein 70 in BRL cells on expression of caspase 3 induced by hydrogen peroxide

目的探讨热休克蛋白（heat shock protein,Hsp）70过表达对过氧化氢（hydrogen peroxide,H_2O_2）诱导Buffalo大鼠肝细胞（BRL细胞）cleaved caspase 3表达的影响。方法将BRL细胞分为6组：空白对照组、Ad-CMV-Null组、Ad-CMV-Hsp70组、H_2O_2组、Ad-CMV-Null＋H_2O_2组和Ad-CMV-Hsp70＋H_2O_2组,Ad-CMV-Null、Ad-CMV-Hsp70、AdCMV-Null＋H_2O_2、Ad-CMV-Hsp70＋H_2O_2组分别加入1×10~7 PFU/ml的Ad-CMV-Null或Ad-CMV-Hsp70培养48 h后,H_2O_2、Ad-CMV-Null＋H_2O_2和Ad-CMV-Hsp70＋H_2O_2组分别加入150μmol/L H_2O_2处理2 h,空白对照、Ad-CMVNull和Ad-CMV-Hsp70组分别加入培养液培养。分别采用实时荧光定量PCR检测细胞中Hsp70基因mRNA的转录水平,Western blot法检测细胞中Hsp70及cleaved caspase 3蛋白的表达水平。结果与H_2O_2组相比,Ad-CMVHsp70＋H_2O_2组细胞中Hsp70基因mRNA的转录水平和蛋白表达水平均显著升高（P〈0.01）,cleaved caspase 3蛋白表达水平显著下降（P〈0.01）。结论Hsp70可抑制caspase 3的激活最终保护BRL细胞,减少凋亡损伤。

Objective To investigate the effect of overexpression of heat shock protein （Hsp） 70 in Buffalo rat liver （BRL） cells on expression of cleaved caspase 3 induced by hydrogen peroxide （H202）. Methods BRL cells were divided into six groups, i. e. blank control, Ad-CMV-Null, Ad-CMV-Hsp70, H2O2, Ad-CMV-Null ＋ H202 and Ad-CMV-Hsp70 ＋ H2O2 groups. The cells in Ad-CMV-Null, Ad-CMV-Hsp70, Ad-CMV-Null ＋ H2O2 and Ad-CMV-Hsp70 ＋ H2O2 groups were added with 1 × 107 PFU/ml Ad-CMV-Null or Ad-CMV-Hsp70 and incubated for 48 h, then those in H2O2, Ad-CMV-Null ＋ H2O2 and Ad-CMV-Hsp70 ＋ H202 groups were treated with 150 μmol/L H2O2 for 2 h, and those in blank control, Ad- CMV-Null and Ad-CMV-Hsp70 groups were added with media and incubated. The transcription level of Hsp70 mRNA in ceils was determined by real-time fluorescent quantitative PCR, while the expression levels of Hsp70 and cleaved caspase 3 by Western blot. Results Comparing with those in H2O2 group, both the mRNA transcription and protein expression levels of Hsp70 in Ad-CMV-Hsp70 ＋ H202 group increased significantly（P 〈 0. 01 ）, while the expression level of cleaved caspase 3 decreased significantly （P 〈 0. 01 ）. Conclusion Hsp70 protect BRL cells from apoptosis by inhibiting caspase 3 activation.