MOLT-4细胞凋亡不同阶段活性Caspase-3在细胞内的空间位相移行

Correlation of Intra-cellular Localization and Variations of Active Caspase-3 to Morphology Changes in Apoptotic MOLT-4 Cells Induced by X-ray

背景与目的:在细胞凋亡过程中,位于细胞浆中的Caspase-3酶原是由什么方式引起细胞形态序贯性变化的问题还没有完全阐明,本研究旨在探讨放射诱导的MOLT-4细胞凋亡不同阶段活性Caspase-3在细胞内的空间分布、细胞形态的变化以及二者的关系。方法:10GyX射线照射诱导MOLT-4细胞。亚G1峰法和凝胶电泳检测凋亡细胞中DNA变化。膜联蛋白V标记细胞膜,带荧光素的Caspase抑制剂(fluorochromeinhibitorofcaspase,FLICA)标记活性Caspase,并用流式细胞仪检测。应用共聚焦显微镜观察MOLT-4细胞凋亡形态及活性Caspase-3在细胞内的分布。免疫印迹检测Caspase-3的表达。结果:X射线10Gy照射后,MOLT-4细胞出现细胞膜翻转、凋亡小体等典型的细胞凋亡特征。其凋亡过程中Caspase-3激活,4h其活性明显增加。在细胞内,活性Caspase-3由靠近细胞膜的胞浆面向细胞浆、胞核移行,与细胞凋亡的形态学变化相吻合,在时间上早于细胞膜翻转。结论:X线照射后MOLT-4细胞中Caspase-3激活,且其亚细胞分布与细胞凋亡的形态学变化相吻合。活性Caspase-3在凋亡细胞中空间位相的移行是放射诱导的细胞凋亡机制之一。

BACKGROUND &OBJECTIVE: How pro-caspase-3 activation lead to ser ia l morphology changes during progress of cell apoptosis is unclear. This study wa s to investigate the variations and intra-localization of active Caspase-3, de termine cell morphology changes in apoptotic MOLT-4 cells induced by X-ray, an d evaluate their relationship. METHODS: MOLT-4 cells were irradiated by 10 Gy X-ray. Sub G1 peak method, and DNA fragmentation assay were used to detect varia tions of DNA in apoptotic cells. Annexin V/PI method was used to determine the c ell membrane reversion, and fluorescence labeled inhibitor of Caspases (FLICA) w as used to detect the active Caspase-3 in apoptotic cells. Cell morphology and Caspase-3 intra-localization were determined by confocal microscopy. RESULTS: MOLT-4 cells irradiated by 10 Gy X-ray presented classical apoptotic morpholog y changes such as membrane reversion, and apoptotic body. Caspase-3 was activat ed after irradiation, and increased remarkably after irradiated for 4 hours. Act ivated Caspase-3 moved from sub-membrane toward cytoplasm and nucleus. Caspase-3 activity was detected 2 hours earlier than membrane reversion. CONCLUSIONS: Caspase-3 was activated in MOLT-4 cells induced by X-ray, and its intra-loca lization correlated with the apoptotic morphology changes. The spatial shift of active Caspase-3 in MOLT-4 cells induced by X-ray is one of the mechanisms of apoptosis.