The Study On The Effect Of Picosecond Pulsed Electric Field On Hela Cells And Their Dose-effect Relationship

Cervical cancer is one of the most common malignant tumor of thefemale reproductive system. In recent years, along with the enhancement ofwomen’s health care consciousness and the expansion and improvement ofcervical cytology and colposcopy examination screening, many patientswith cervical cancer and precancerous lesions can be detected and treatedearly. At present, the age of cervical cancer patients is younger year by year,while the traditional surgical treatment can often damage the reproductiveorgans severely, which influence their fertility and sexual function badlyand is a serious threat to the patients, especially the young patient s ’physical and mental health. Although, the idea of surgical treatment ischanging from traditional way to guarantee the curative effect andminimize tissue trauma at the same time so as to preserve the fertilityfunction. But this operation method has its strict indications, andpostoperative pregnancy rate is low, the risk of miscarriage and prematurebirth after pregnancy is high. Facing this new problem, we hope to find anew, safe and noninvasive treatment that can not only kill the tumor cellseffectively, but also reduce or even no damage to reproductive organs so asto preserve their reproductive and sexual function.Pulsed Electric field (PEF) can significantly affect the cellmorphology and signal transduction and then cause cells to reversibleelectroporation (RE), irreversible electrical breakdown(IREB) andintracellular electromanipulation (IEM） due to its special physicalproperties. PEF is gradually developed to a new type of tumor treatments. However, Microsecond or Nanosecond pulsed electric fields need electrodearray of a minimally invasive needle to insert into the tumor tissue, whichwill undoubtedly bring inconvenience to operation, some discomfort topatients and may cause the metastasis of the tumor cell at the same time,etc. To some extent, these limit the application of this method in clinic.When the pulse electric field amplitude rises to hundreds or even thousandsof kV/cm and the pulse width reduced to picosecond level (ps), it has richultra broadband frequency spectrum. Time and spatial resolution are highwhile signal distortion is low. Chongqing university, Our experimentalcooperation unit, has developed a ultra-wideband impulse radiating antenna(IRA), which is composed of the ellipsoid reflector and single cone radiator.It can transfer energy in a specific range, converge the pulseelectromagnetic wave in the designated area and destroy the target.Radiation energy is highly concentrated both in time and space. Based onthe optical principle, psPEF can be transferred to the target position of deepbiological tissue without trauma and focused effectively by IRA. In thisway, psPEF could kill tumor cells and avoid damage to normal tissuesaround at the same time, so that noninvasive treatment for tumors ispromising. In2011, our cooperator improve performance of picosecondpulse generator. The measured results showed that the the electric field ofpicosecond pulse generator is up to600kV/cm and the width is800ps withstable discharge, good repeatability, which makes it meet the requirementsof IRA. Nevertheless, research on the biological effects induced by psPEFin tumor cells is reported rarely. In recent years, Our experimental grouphas done some researcheS on the biological effects induced of psPEF so asto get suitable parameters of psPEF that can induce the killing effect ontumor cells, so as to lay a foundation for subsequent experiments in vivoand clinical trials. In this study, we exposed HeLa cells to different psPEF conditions.MTT assay, flow cytometry and transmission electron microscopy werethen used to detect the effects of psPEF on HeLa cell growth inhibition,cell apoptosis, and cell cycle. Meanwhile, we further discussed therelationship between the psPEF and biological effect of HeLa cells throughchanging the psPEF parameters. In this study, we hope to get suitableparameters of psPEF that can induce the killing effect on tumor cells, so asto lay a foundation for subsequent experiments in vivo and clinical trials. Inthis way, noninvasive treatment that can keep reproductive function foryoung cervical cancer patients would be realized.Part1The effect on cell proliferation of HeLa cells inducedby psPEF and its dose-effect relationshipObjective: to study the effect of psPEF on proliferation of HeLa cellsand its dose-effect relationship.Methods: Cells of experiment groups were exposed to800psecpulses, a frequency of3Hz,2000pulse number and different electric fieldamplitudes (100,200,300,400,500and600kV/cm). And then each groupwas routinely cultured for6,12,24,36and48h, the inhibition rate of cellproliferation was then determined by MTT method.According to the results determined by the above MTT experiment,the culture time after psPEF treatment were all12h in the subsequent trials.The test were divided into6groups according to electric field amplitude(100,200,300,400,500and600kV/cm), with each group being furtherdivided into several subgroups according to pulse number (1000,2000and 3000). The other parameters of each group were the same (800psec pulses,a frequency of3HZ). the inhibition rate of cell proliferation was thendetermined by MTT method. Due to the cell proliferation inhibition in theexperiment results of200kV/cm,400kV/cm and600kV/cm group isespecially obvious, we chose the electric field amplitude of200kV/cm,400kV/cm,600kV/cm as low-middle-high group in the following tests.According to the experimental results above, we exposed HeLa cellsto2000pulse number,800psec pulses and various electric field amplitudes(200,400, and600kV/cm) at a frequency of3Hz. After12h incubation,we measured distribution of cell cycle with a FACS Vantage SE flowcytometer.Results: After psPEF treatment, compared with the control group, thecell proliferation of each experiment group was inhibited significantly (P <0.05). Compared to6h groups, the growth inhibition rates of HeLa cells in12h group was very notable (P<0.01). However, the comparitions ofgrowth inhibition rates between12h and24h groups were not obvious (P＞0.05). No significant differences (p>0.05) were also noted between the24hgroup and36h groups,36h groups and48h groups (p>0.05). while electricfield amplitude was given the same, with the pulse number getting more,the growth inhibition rates becomes higher (P<0.05). Similarly, in eachnumber group (from1000to3000group), with the amplitude gettinghigher, the cell growth inhibition rate increased significantly (P<0.05). thegrowth inhibition rates was the highest when the electric field amplitudewas600kV/cm and pulse number was3000.In the detection of cell cycle progression of HeLa cells after psPEFtreatment, the ratios of G2/M phase of the cell cycle in all three experimentgroups were more than that in untreated group (P<0.05). As the amplitude increased, the number of G2/M phase cells rised significantly, while thenumber of cells in the G1phase decreased gradually.Conclusions: A certain dosage of psPEF can inhibit HeLa cellproliferation, and the cell cycle is arrested in G2/M phase. cell growthinhibition reaches the maximum after12h incubation after psPEFtreatment. The effect of cell growth inhibition of psPEF is Positivelyrelated to electric field amplitude and pulse number.Part two The effect on cell apoptosis and necrosis of HeLacells induced by psPEF and its dose-effect relationshipObjective: to study the effect of psPEF on cell apoptosis and necrosisof HeLa cells and its dose-effect relationship.Methods: According to the results of the first part. groups are asfollows: according to different psPEF electric field amplitude, we dividedinto HeLa into4groups: untreated group and3different electric fieldamplitude group (200kV/cm group,400kV/cm group, group600kV/cm),other parameters were fixed to800ps pulse width,3Hz of frequency and2000of pulse number. After12h incubation, we used flow cytometry andtransmission electron microscopy were to detect the effects of psPEF oncell apoptosis and necrosis. According to different pulse number, HeLacells were divided into4groups: control group and3different pulsenumber (1000group,2000group,2000group), other parameters werefixed to800ps pulse width,3Hz of frequency and400kV/cm of electricfield amplitude. After12h incubation, we used flow cytometry to detectthe effects of psPEF on cell apoptosis and necrosis.Results: After psPEF treatment, we saw typical apoptotic cells in thethree treatment groups. the apoptosis rates in1000,2000and3000pulsenumber group were much higher (P<0.05) than that in the the untreated group. Meanwhile, compared to the control group, the apoptosis rate of200,400and600kV/cm group increased significantly (P<0.05). With theelectric field amplitude and pulse number getting higher, the apoptosis ratesincreased. However, there was on significant difference (P>0.05) in thecomparation of cell necrosis rate between two close groups.Conclusions: a certain dosage of psPEF can induce apoptosis of HeLa,and the effect is positively related to the electric field amplitude and pulsenumber.