The Change Of Mitochondrial And Calcium During The Apoptosis Induced By SfaMNPV

Many researches were carried through in mammal animals showed that mitochondrial an ER might have changed much after apoptosis induced by a variety of stimuli, including cytokines, anti-cancer drugs, growth factor deprivation and so on. However, few researches were gone on the apoptosis in the insect cells. In this work, effects of SfaMNPV on mitochondrial and ER in SL-1 cells were studied. The main results were as follows:Effect of SfaMNPV(MOI=6) on the function of mitochondrial in SL-1 cells was investigated by MTT assay and membrane potential was assayed by both flow cytometry and confocal laser scanning microscope. The results showed that the function of mitochondrial began to decline after induction for 8h, and the declining rate was typical in time-dependent manners. Under the flow cytometry assay, the effect of SfaMNPV on mitochondrial membrane potential(△Ψ m) was investigated by Rh-123 staining of cells. The Rh-123 fluorescent intensity in SL-1 cells was reduced in time-dependent manner. The results of confocal laser scanning microscope confirmed that SfaMNPV was able to reduce AWm. The effects of SfaMNPV on apoptosis-related protein Bcl-2 and cyto c were investigated by western blotting. SfaMNPV could down-regulate the level of Bcl-2 expression after induction for 8h.Also,cyto c was released from mitochondrial to cytosolic after induction for 4h.ER is well characterized as a mobilizable calcium store that sequesters excess cytosolic calcium and a reservoir for calcium signaling to maintain intracellular Ca2+ homeostasis. Effect of SfaMNPV on [Ca2+]i of SL-1 cells was studied by observing fluorescent intensity variation of cells stained with fluo-3/am using confocal laser scanning microscope. The results showed that [Ca2+]i was rapidly increased after cells were induced by SfaMNPV. The fluo-3 fluorescent intensity was 3-fold of that of not induced cells after SfaMNPV induction. When removing calcium from the medium by compound EGTA to medium, [Ca2+]i response toSfaMNPV was inhibited completely. These results suggested that extracellular calcium ion influx is the main source of intracellular calcium increase .To determine which factors might serve as the primary mediator for baculovirus SfaMNPV-induced SL-1 cells apoptosis, the effects of EGTA and the clamped cytosolic Ca2+ condition on baculovirus-induced apoptosis were studied. Having established certain conditions by culturing SL-1 cells in Grace medium with 7PM BAPTA/AM and 110MM CaCL;,which sustained a cytosolic Ca2+ clamp yet did not depleted intracellular Ca2+ stores and induced apoptosis, the apoptosis in SfaMNPV-infected SL-1 cell was detected. The results showed the condition could not prevent apoptosis in SL-1 cells. Taken together,these data suggested that the depletion of ER Ca2+ stores rather than the elevation of cytosolic Ca2+ or extracellular Ca2+entry contributed to SfaMNPV-induced SL-1 cells apoptosis. This result was the same to many researches.