论文部分内容阅读
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: The question of the manner in which caspase-3 zymogen is located in the cytoplasm during cell apoptosis is not fully elucidated in what manner. The aim of this study was to investigate the role of radiation-induced MOLT-4 The spatial distribution of active Caspase-3 in different stages of cell apoptosis, the change of cell morphology and the relationship between the two. Methods: MOLT-4 cells were induced by 10 Gy X-ray irradiation. Sub-G1 peak and gel electrophoresis were used to detect DNA changes in apoptotic cells. Annexin V labeled cell membrane, fluorescein inhibitor of caspase (FLICA) labeled active Caspase and detected by flow cytometry. Confocal microscopy was used to observe the apoptotic morphology of MOLT-4 cells and the distribution of active Caspase-3 in the cells. Western blot was used to detect the expression of Caspase-3. Results: The apoptosis of MOLT-4 cells was observed after 10Gy X-ray irradiation, such as cell membrane turnover and apoptotic bodies. The apoptosis of Caspase-3 activation, 4h its activity was significantly increased. In the cell, active Caspase-3 from the cytoplasm near the cell membrane toward the cytoplasm, nucleus migration, consistent with the morphological changes of apoptosis, in time earlier than the cell membrane turnover. CONCLUSION: Caspase-3 is activated in MOLT-4 cells after X-ray irradiation, and its subcellular distribution coincides with the morphological changes of apoptosis. The migration of active Caspase-3 in the spatial phase of apoptotic cells is one of the mechanisms of radiation-induced apoptosis.