Research On Damaging Effect Of HeLa Cells Induced By Intense Picosecond Pulsed Electric Field

Cervical cancer is one of the most pervasive genital tract malignanttumors in female, which is second to breast cancer. Recently, with thedeveloping of the cervical cancer detection level and popularizing ofscreening system, the incidence of cancer detection rised obviously andyounger patients becoming more and more, which is a serious threat towomen’s health and lives. At present, the main treatment to cervical canceris surgery, assist with radiotherapy, chemotherapy and so on. Cervicalcancer patients may be cured by resection at the very earlier stage, however,decrease in the quality of life and increase in the incidence of abortion andpremature delivery appear, sometimes they even lose the ability ofreproductively. Therefore, new treatments are needed because of the poortherapeutic effects of traditional treatments.Pulsed power technology, which combines biomedicine engineering,high voltage engineering and microelectronics, have been emerging as anew technology in clinical tumor treatment. The cervical cancer can betreated by pulsed electric field easily because of easy exposure and fixedposition. Intense picosecond pulsed electric field (psPEF) was deemed to bethe good choice for noninvasive treatment due to its physical characteristicsof higher electric intensity and narrower pulse width, which hasextraordinary concentricity, high excitation energy, abundant ultra wideband frequency spectrum, accurate direction and easy to be controlled. By now, there were rarely seldom researches about intense psPEFdomestic and overseas. Our research group for the first time to study thedamage effects and mechanisms of intense psPEF interiorly, aimed tooffering basic researches for the noninvasive treatment,which was based ontargeted apoptosis induction.PART ONEBRIEF INTRODUCTION OF THE INTENSEPICOSECOND PULSE GENERRATORWith impulse radiating antenna, psPEF could get into the biologicaltissues non-invasively and specifically.State Key Laboratory of PowerTransmission Equipment and System Security and New Technology inChongqing University developed intense psPEF through front steeping andtail cutting in the Marx generator, which generated nanosecond pulses. Theexperimental results indicate that the psPEF can produce pulses withamplitude of50.0kV, pulse duration of about800ps and the rising edge ofno more than250ps, stable and reproducible discharge, which can meet therequires of the impulse radiating antenna.The intense picosecond pulser canprovide solid foundation for the non-invasive treatment based on targetedtumor cell apoptosis.PART TWOTHE DOSE-EFFECT RELATIONSHIP OF GROWTHINHIBITION IN HELA CELLS INDUCED BY INTENSEPICOSECOND PULSED ELECTRIC FIELDObjective: To explore the dose-effect relationship between the dosage of intense psPEF and the growth inhibition in HeLa cells in vitro.Methods: Intense psPEF with constant parameters (pulse duration of800ps, repeat frequency of3Hz) and different dose of electric pulses(150kV/cm,1000pulses;150kV/cm,3000pulses;250kV/cm,1000pulses) wereperformed on HeLa cells. Trypan blue staining was used for calculating cellmortality after the cells were treated for0h,6h,12h,18h,24h,36h and48h;High-intensity psPEF with duration of800ps, frequency of3Hz anddifferent electric intensities (50kV/cm,150kV/cm,250kV/cm) performedon HeLa cells. Every electric intensity group with the pulses of0,100,500,1000,2000,3000,4000and5000. MTT assay was used to trace theeffect of growth inhibition at6h and12h.Results: Intense psPEF could obviously effect the growth of HeLacells and show the character of time-dependent and dose-dependent. Themaximum inhibitory rate was observed at12h after the treatment, there is nostatistical difference between the groups after12h(P>0.05);Intense psPEFwith250kV/cm,5000pulses has the maximal inhibition ratio.Conclusion: Intense psPEF in a certain range can inhibit the growth ofHeLa cells.PART THREEOBSERVATION OF THE DAMAGING EFFECT OF HELACELLS INDUCED BY INTENSE PICOSECOND PULSEDELECTRIC FIELDObjective: To observe the damage effect of HeLa cells induced byintense psPEF.Methods: Intense psPEF with constant parameter (pulse duration of 800ps, frequency of3Hz, electric intensity of250kV/cm) and differentpulses(0,1000,3000,5000) were performed on HeLa cells. At12h after thetreatment, the morphology change of HeLa cells were detected bytransmission electron microscope and AO/EB dye. Immune cell chemicalS-P method was used to detect the expression of caspase-3; the expressionof Bcl-2/Bax were detected by Western Blot.Results: The typical changes of apoptosis were observed bytransmission electron microscope and AO/EB dye. The expression ofcaspase-3in treatment groups was higher than control group (p﹤0.05), andthe expression of caspase-3increased along with the dose added. Theexpression of Bax in treatment groups was obviously higher than that incontrol group（p﹤0.05）, however Bcl-2was lower, the Bax/Bcl-2increased obviously（p﹤0.05）.Conclusion: Intense psPEF can induce HeLa cells apoptosis.