半胱氨酸天冬氨酸3依赖的bcl-2的降解在ATP耗竭诱导肾小管上皮细胞凋亡中的作用

目的 研究ATP耗竭再恢复诱导的肾小管上皮细胞凋亡中,半胱氨酸天冬氨酸（caspase）3激活介导bcl-2降解的作用.方法 使用代谢抑制剂短暂阻断细胞内ATP的生成,诱导细胞凋亡.以Hoechst 33342染色观察凋亡细胞.应用编码人bcl-2 RNA的腺病毒感染细胞,使bcl-2在细胞内高表达.应用流式细胞仪,分析细胞凋亡和坏死的发生情况.以Western印迹检测caspase 3的活化以及bcl-2和bcl-2的降解产物.体外实验观察caspase 3和caspase 3特异性抑制剂DEVD对bcl-2降解产物的影响.结果 高表达bcl-2组与对照组比较,细胞ATP耗竭60min、90 min、120 min和再恢复60min后,可减少细胞凋亡50%,P＜0.05.肾小管上皮细胞ATP耗竭时,caspase 3被激活,bcl-2出现降解;细胞内ATP恢复时,bcl-2降解产物继续增多,并出现细胞凋亡.体外应用caspase 3可使bcl-2降解,而caspase3的特异性抑制剂DEVD能明显抑制caspase 3对bcl-2的降解.结论 肾小管上皮细胞ATP耗竭再恢复时,caspase 3激活及其介导的bcl-2降解在细胞凋亡中发挥重要作用.

The role of caspase 3-dependent bcl-2 degradation in ATP depleted apoptosis in renal epithelial cells

Objective To investigate the role of caspase 3 in ATP depleted apoptosis in renal tubular epithelial cells (RTEC). Methods To induce apoptosis, REC were subjected to 60 min ATP depletion followed by recovery. Wild-type bcl-2 was overexpressed by infecting opossum kidney (OK) cells with adenovirus containing wild-type human bcl-2. Apoptotie cells were detected with Hoechst 33342 dye. Flow cytometry was used to quantify apoptosis and necrosis. Active caspase 3 and bcl-2 degradation were assessed by Western blot. Results ATP depletion and recovery resulted in activation of caspase 3 and the progressive accumulation of bcl-2 cleavage products and apoptosis. Overexpression of bcl-2 ameliorated apoptosis in the ATP-depleted RTEC followed by recovery. Caspase 3 reproduced the effect of caspase 3 on bcl-2 cleavage, whereas caspase 3 specific inhibitor DVED prevented bcl-2 cleavage in vitro. Conclusion Caspase 3 activition and mediated-bcl-2 degradation are likely to contribute to ATP depletion-induced apoptosis in RTEC.