前凋亡因子Bim在地塞米松诱导小鼠胸腺细胞凋亡中的作用

Role of Pro-apoptotic Protein Bim in Mouse Thymocytes Apoptosis Induced by Dexamethasone

目的研究前凋亡因子Bim在地塞米松诱导小鼠胸腺细胞凋亡中的作用。方法体外培养BALB/c小鼠胸腺细胞,终浓度1μmol/L地塞米松诱导细胞,透射电镜、AnnexinV/PI双染流式细胞术、DNA凝胶电泳检测细胞凋亡;RT-PCR半定量法分析1μmol/L地塞米松作用不同时间以及不同浓度地塞米松作用1 h小鼠胸腺细胞Bim mRNA表达。结果11μmol/L地塞米松作用4 h小鼠胸腺细胞出现特征性DNA梯状条带和细胞凋亡典型形态特征,流式细胞术检测显示在不同时间点(2 h,4 h,8 h)实验组细胞凋亡率较对照高;21μmol/L地塞米松作用1/2 h后小鼠胸腺细胞Bim mRNA三个异构体表达即明显升高,以BimL最为明显,其中BimL、BimELmRNA表达在1 h达高峰,较对照组高峰前移11 h,且各时间点(0 h、24 h除外)表达水平与同时点对照组相比差异具有统计学意义(P<0.05);BimS mRNA表达高峰较对照组提前4 h,但各时间点表达水平与同时点对照组相比差异无统计学意义(P>0.05);3不同浓度(0,10-2,10-1,1,10μmol/L)地塞米松作用胸腺细胞1 h时,其活细胞率和凋亡细胞百分率在不同浓度组间的差异无统计学意义,而Bim mRNA表达随地塞米松终浓度增加而升高,也以BimL mRNA表达增高最明显。结论Bim参与了地塞米松诱导的小鼠胸腺细胞凋亡过程,Bim三个异构体中以BimL作用为主;Bim mRNA表达发生在细胞凋亡早期,其表达水平随作用时间变化,并在一定地塞米松浓度范围内呈剂量依赖性。

Objective To explore the role of pro-apoptotic protein Bim in the mouse thymocyte apoptosis induced by dexamethasone（DEX）.Methods 1 μmol/L of dexamethasone was used to induce the apoptosis of BALB/c mouse thymocyte cultured in vitro.The thymocyte apoptosis was detected by transmission electron microscope,Annexin V-FITC/PI flow cytometry and DNA agarose gel electrophoresis.The RT-PCR method was applied to the semi-quantitative analysis of Bim mRNA expression induced by 1 μmol/L DEX at different time point and by different final concentration dexamethasone used for 1 h.Results ① With 1 μmol/L DEX used to cell incubation for 4 h, the mouse thymocyte showed the morphological characteristics of apoptosis and the typical DNA ladder on electrophoresis gel.The analyses of Annexin V-FITC /PI flow cytometry showed that the thymocyte apoptosis rates at different time point（2 h,4 h,8 h） were significantly higher in dexamethasone treated group than in control group.② With 1 μmol/L DEX used to cell incubation for 1/2 hr,the expressions of three Bim mRNAs in mouse thymocyte began to increase and the BimL expression was predominant among three mRNA isoforms.The peak values of BimL and BimEL mRNA expression appeared at 1 h and 11 h which were earlier to dexamethasone treated group than control group,and the BimL and BimEL expression levels at different time point（except at 0 h and 24 h） were significantly increased（P〈0.05）.The peak value of BimS mRNA expression of dexamethasone treated group was 4 hours earlier than that of control group,but there was no significant difference between two groups（P〉0.05）.（3） When the mouse thymocyte was incubated with various concentration（0,10^-2,10^-1,1,10 μmol/L） DEX for 1 h,there was no significant percentage difference occurring between living and apoptotic cell.While the total of Bim mRNA expression was increasing in a concentration-dependent manner,the mRNA increase of BimL isoform was still the most predominant among three mRNA isoforms.Conclusion The pro-apoptotic protein Bim plays an important role in mouse thymocyte apoptosis induced by dexamethasone,and the BimL mRNA is the predominant isoform in this process.The increase of Bim mRNA expression appears in very early stage of thymocyte apoptosis and both time-and dose-dependent.