Influence Of Cytochrome C On Apoptosis Of Spodoptera Litura Cells In Cell-free System

Apoptosis is one of the hot research topics of the life science.Many new methods and techniques have emerged with the scientific and technological development.They have provided powerful weapons for the study of apoptosis.Cell-free system has irreplaceable advantages at the application of apoptosis.It is mainly used in the sub-cellular and molecular level studies of the reconstruction of the nucleus in vitro,cell cycle regulation,DNA replication and other mechanisms of cell life activities.Cell-free system was applied to apoptosis the first time in 1993 by Lazebnik.And the results were great in cell-free system of mammalian and botanical cells.But the plication of apoptosis is being researched deeply in insect cell-free system.In this paper,apoptosis of insect nuclei have been concerned to explore and study in insect cell-free system.The results of our previous studies have proved:it is a key step in SfMNPV-induced and AcMNPV-induced apoptosis in Spodoptera litura line(SI-1) to release cytochrome c (cytc) from the mitochondria to the cytoplasm.The nuclei of the Sl-lcells are our studying objects in this experiment,cell-free system is prepared by ultracentrifugation of Sl-1 plasma and activated by cytc.We studyed the characteristics of apoptotic nuclei,and further explored the effects of cytc on nuclei of insect cells.The results were as the follows:(1) the effects of exogenous cytc on the nuclei of Sl-1 cellsThe morphology of apoptosis is the classic standard of apoptosis detection. Fluorescent microscope was applied to observe the apoptotic process of nuclei dyed by DAPI.It is the most useful and the most easy way.Activated apoptosis-specific nuclease specifically cuts the chromatin into a DNA ladder between the nucleosomes.The DNA ladder of agarose gel eletrophoresis could be seen clearly.The nuclei of Sl-1 cells presented typical morphologic changes and DNA ladder formation were observed. Results were as the follows:the prepared cell-free system was shown to be activated by cytc.Nuclei were incubated for different hours at 28℃in the cell-free induction system. After incubation for 2 hours in the induction system,the chromatin in the nuclei underwent condensation and margination.After incubation for 4 hours in the induction system,vesicles with a small amount of condensed chromatin were expelled from the nuclei.And apoptotic bodies containing condensed chromatin were seen being expelled from the nuclei after incubation for 6 hours.After incubation for 8 hours,the apoptotic nucleus was split into several apoptotic bodies.But chromatin in the nucleus added into the cell-free system without cytochrome C remained intact after 12 hours incubation.These results showed that cytochrome C could induce apoptosis of nuclei.DNA ladder could be observed after the nuclei had been induced in the induction system.And the total DNA of nucleus was not degraded after incubation for 12 hours in the cell-free system without cytochrome C.(2) the effects of exogenous cytchrome c on the caspase-3 activityThe central component of apoptosis machinery is a group of cystein proteases called caspases that cleave proteins after aspartic acid residues.Procaspases are activated during apoptosis.The activity of caspase has become an important indicator of apoptosis detection.An activation of caspase-3 in cytchrome c-induced cell-free induction system was determined in AC-DEVD-AFC as substrate.At 4h post induction an activity of caspase-3 was measured and gradually increased with time extension.These data indicate that cytchrome c play a key role in cytchrome c-induced cell-free induction system in Spodoptera litura cells.