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Cancer is one of the major diseases that threaten human health, but the ideal treatment option is still a worldwide problem in recent years nanosecond steep pulse-induced apoptosis of tumor cells for the treatment of tumor lesions, showing exciting clinical application. Studies have shown that when the electric field pulse width is reduced to the level of nanoseconds (ns), the electric field strength increases to tens of kV / cm, a series of functional and structural changes in the interior of the cell, a phenomenon known as the intracellular processing effect (Intracellular Apoptotic cells under certain physiological or pathological conditions, to follow its own procedures to the end of the process of life, plays a key role of mitochondria in apoptosis signal transduction pathway [3]. This experiment proceed from the mitochondrial apoptotic pathway the capsule gland serous human ovarian cancer SKOV3 cells as experimental subjects, further study nanosecond steep pulse induced apoptosis of tumor cells. The experiment is divided into two parts: the nanosecond steep pulse on the SKOV3 cells induce apoptosis and best steep pulse parameters; 2, nanosecond steep pulse induced the SKOV3 cell apoptosis process mitochondrial function and apoptosis-related molecules Expression change. 1 nanosecond steep pulse on the SKOV3 cells induce apoptosis and best steep pulse parameters 1.1 Materials and methods 1.1.1 field strength 50KV/cm pulse width of 50ns, 100ns, 200ns, the number of pulses were 10,30,60,80 steep pulse processing SKOV3 cells for the experimental group, the SKOV3 cells untreated as a control, application of Annexin V / PI double staining, flow cytometry detection of early apoptosis rate; agarose gel electrophoresis assay were incubated with the DNA ladder 6h post-treatment group and control group. 1.1.2 at 50kV/cm, 100ns, 30 pulsed electric field processing SKOV3 cells were incubated for 30 min, 2h, 6h and 12h after extraction of total protein was detected by Western blot method Caspase-3 active fragment. 1.2 Results 1.2.1 compared with the control group, nanosecond steep pulse treated Annexin V / PI double staining flow cytometry results showed that the pulse width of less than 100ns the Annexin V ~ / PI to - mainly phosphatidylserine (PS) valgus, maintain the integrity of the cell membrane, prompted pulsed electric field the SKOV3 cells within the treatment effect; pulse width of 200ns, Annexin V / PI, PI penetrate the cell membrane into the nucleus, cell membrane rupture, suggesting that Pulsed Electric Field on SKOV3 cells by electroporation role-based. DNA agarose gel electrophoresis showed that the pulse width 50ns, 100ns nanosecond the steep pulse processing group has a large number of DNA ladder formation, prompt SKOV3 cells apoptosis; 200ns treated strip film paste no obvious DNA ladder formation, suggesting that The SKOV3 cell necrosis. 1.2.2 Compared with the control group, treated SKOV3 cells Caspase-3 active fragment the expression of a significant increase (P <0.05). 1.3 Conclusion nanosecond steep pulse dealt with the role of cells can induce apoptosis of SKOV3 cells in vitro, the optimum pulse width of 50 to 100ns minimum field strength 50kV/cm apoptosis rate in a certain number of pulses, with pulse The increase in the number increased to continue to increase the number of pulses, apoptosis rate increased mortality. 2 nanosecond steep pulse SKOV3 cell apoptosis induced by altered expression of mitochondrial function and apoptosis related molecules. 2.1 Materials and Methods 2.1.1 50kV/cm, 100ns the 30 steep pulse processing SKOV3 cells were incubated for 30 min, 2H, 6h, 12h after the treatment group and the control assembly load reactive oxygen fluorescent probe DCFH-DA streaming cytometric detection of different time periods SKOV3 cells mitochondrial reactive oxygen species (ROS) changes. 2.1.2 50kV/cm, 100ns, 30 steep pulse processing SKOV3 cells were incubated for 30 min, 2H, 6h, 12h after the treatment group and the control assembly loaded membrane potential fluorescent probe JC-1 flow cytometry mitochondria membrane potential changes (△ Ψ_m). 2.1.3 50KV/cm, 100ns, 30 steep pulse SKOV3 cells, immunofluorescence detection incubated for 6h after the treatment group and the control group the mitochondrial membrane space protein cytochrome-C, AIF distribution changes; Western blotting method detection were incubated for 30 min, 2 h , 6h, 12h after the treatment and control groups SKOV3 cytoplasmic cytochrome-C, AIF expression level changes. 2.1.4 50KV/cm, 100ns, 30 steep pulse processing of SKOV3 cells, western blot method of apoptosis regulatory proteins Bax and Bcl-2 expression levels of change. 2.2 Results 2.2.1 nanosecond steep pulse after SKOV3 cells compared with the control group, the fluorescence intensity of intracellular ROS immediately increased (P <0.05), with time, the fluorescence intensity diminished fluorescence intensity after 12 hours lower than the control group. 2.2.2 Compared with the control group, SKOV3 cells treated by nanosecond steep pulse 2h △ phimax began to decrease to a minimum at 6h (P <0.05), continued to 12h the △ Ψ_m still lower than the control group (P < 0.05). 2.2.3 Compared with the control group, SKOV3 cells treated by nanosecond steep pulse cytochrome C, AIF released by the mitochondrial membrane gap were localized in the cytoplasm, the nucleus, cytoplasmic cytochrome C, AIF expression increased (P <0.05). 2.2.4 compared with the control group, the expression of pro-apoptotic protein Bax nanosecond steep pulse after SKOV3 cells, a significant increase (P <0.05) inhibition of apoptotic protein Bcl-2 expression is slightly reduced (P> 0.05), but Bcl-2/Bax significantly reduced (P <0.05). 2.3 Conclusion nanosecond steep pulse-induced apoptosis of SKOV3 cells, mitochondrial function and apoptosis-related molecules altered expression and steep pulses in the nanosecond range, mitochondria may play an important role in the induction of apoptosis.
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