The Dection Of Apoptosome In Sl-1 Cells Deduced By AfMNPV

Apoptosis is a kind of independent, orderly and according to some scheduled program of natural death process. Apoptosis is one of the most popular field in life science. Many related stimuli coming from intracellular or extracellular result in the releasing of cytochrome c. Then the cytochrome c quickly combines with the apoptotic protease activating factor-1(Apaf-1), ATP/dATP and procaspase-9, which forms a multimeric protein complex (-700 kDa) called apoptosome. The activation of Caspase-9 subsequently triggers a cascade of effector caspase activation,leading to apoptotic cell death. It's very improtant for mitochondrial signal pathway and embryonic development in mammal. In this paper, the existence of apoptosome of insects has been preliminary researched.Virus is a common induction facor of apoptosis. In this study, Sl-1 cells were induced by AfMNPV which is a kind of baculovirus. DAPI stain, agarose gel electrophoresis, fluorescence quantitative, and Western Blotting have used to detect the characteristics of morphology and biochemistry, the activity of Caspase-9, and the existence of apoptosome respectively.The DAPI stain and agarose gel electrophoresis indicated that cell apoptosis have happened afer 8 h which induced by AfMNPV. Caspase-9 will be activate first, once the apoptosome has formed. Therefore, we use the specific fluorescence substrate (Ac-LEHD-AFC) and specific inhibitors (Z-LEHD-FMK) of Caspase-9 to detect the activity of Caspase-9. The results show that the activity of Caspase-9 was measured afer 8 h which induced by AfMNPV, and the activity increased to the max at 24 h. In order to detect the existence of apoptosome, we use Western Blotting to analyze the protein component of apoptosome. The proteins that compose the complex can be detected afer 6 h which induced by AfMNPV, and then they disappeared at 12 h. These above results preliminary show that, as mammalian cell apoptosis, insect cell apoptosis alos have the apoptosome formation.