Studies On Apoptosis Of Human Ovarian Carcinoma Cell Line SKOV3 In Vitro And Its Mechanism By Nanosecond Steep Pulse

The pulsed electric fields (PEF),due to its unique electrobiological effect,has showed good prospective application for tumor therapy.Futher research found that Intracellular changes very different from electroporation phenomenon could be induced by nanosecond pulsed electric fields (nsPEF).Compared with the traditional electroporation,this phenomenon is termed intracellular electromanipulation (IEM).nsPEF can penetrate cell interior due to its high equivalent frequency, leading to a series of functional changes in nucleolus, mitochondria,endoplasmic reticulum,and cell signal transfer,which provided possible foundation of apoptosis under nsPEF.Nowadays,experiment and mechanism research on cancer cell apoptosis induced by nsPEF have become a research hotspot in the field of tumor therapy internationally.But in these articles reserch on the apoptotic morphological feature under nsPEF was less,and disease entities were limited.in addition,the exact mechanism of apoptosis induced by nsPEF remains to be studied furtherly.So in this study,we plan to investigate the apoptotic morphological and functional changes,in order to ascertain the mechanism of apoptosis induced by nanosecond steep pulse on human ovarian carcinoma cell Line SKOV3 in vitro.We hope to provide a theoretical and experimental basement for the clinical application of nanosecond steep pulse therapy. PART I:GROWTH-INHIBITED AND APOPTOSIS-INDUCED EFFECTS OF NANOSECOND STEEP PULSE ON HUMAN OVARIAN CARCINOMA CELL LINE SKOV3Objective:To investigate the effect of Nanosecond Steep Pulse on growth and apoptosis in human ovarian carcinoma cells in vitro.Methods:After SKOV3 cells were treated with nanosecond steep pulse of 8.5,10,12.5 kilovolts per centimeter(kV/cm) amplitude respectively,MTT assay,invert microscopy,fluorescence microscopy and AnnexinV-FITC were performed to indentify the inhibitory effect of nanosecond steep pulse on cell proliferation and the induction of apoptosis in human ovarian carcinoma cell Line SKOV3.Results:Nanosecond steep pulse with 10 and 12.5 kV/cm significantly inhibited the viability of SKOV3 cells in a dose-dependent manner,and in a time-dependent manner within the range of 2 h to 4 h.The typical changes of SKOV3 cell apoptosis were observed by invert microscope and fluorescence microscopy.Early apoptotic rates of SKOV3 treated with 10,12.5 kV/cm nanosecond steep pulse were(22.21±2.71)% and(57.30±6.51)%, respectively,significantly higher than those of the control group(3.04±0.44)% (P<0.05).Conclusion:Nanosecond steep pulse in a certain range can inhibite the growth of SKOV3 cells as well as induce the apoptosis. PART II:ELECTRON MICROSCOPIC OBSERVATION AND MECHANISMS STUDIES ON APOPTOSIS OF HUMAN OVARIAN CARCINOMA CELL LINE SKOV3 INDUCED BY NANOSECOND STEEP PULSEObjective:To investigate the apoptotic morphological features and changes of the intracellular calcium concentration ([Ca²⁺]i ) induced by nanosecond steep pulse on human ovarian carcinoma cell Line SKOV3 in vitro.Methods:SKOV3 cells were exposed to nanosecond steep pulse (10kV/cm,100ns,1 Hz in 5 minutes). At 4h after treatment, the apoptosis of SKOV3 cells was detected by transmission electron microscope and scanning electron microscope.The change of [Ca²⁺]i in SKOV3 cells and the source of Ca²⁺ during [Ca²⁺]i change were observed by laser scanning confocal microscope using Fluo-3/AM as the calcium fluorenscent indicator.Results:The typical morphological features of apoptotic cells were observed by transmission electron microscope and scanning electron microscope.The [Ca²⁺]i was markedly increased by treatment with nanosecond steep pulse(P<0.01),while the increase showed no relationship to the extracellular calcium concentration(P>0.05). Conclusion:Nanosecond steep pulse can induce apoptosis of SKOV3 cells.The increase of [Ca²⁺]i through release of intracellular calcium might be one of the mechanisms of the apoptosis.