The Effect Of Low Field And High Frequency Nspefs On The Biological Behavior Of Human Melanoma Cells

Melanoma is a malignant tumor with a high degree of malignancy,the average age of onset is about 45 years old,and the incidence of the disease increases with age.There is still a lack of effective means for melanoma treatment.The pulsed electric field(pulse electric fields,PEFs)was made on the basis of electroporation technology which has become a breakthrough in the search for new ways to treat cancer.Especially,the pulse width of nanosecond pulsed electric field(nano-second pulse electric fields,nsPEFs)which is a kind of instantaneous release of pure and very high energy physical therapy,it is non-toxic,no heat,no side effect,short treatment duration.Low field(<10 kV/cm)electric field compared with the current high field of strong high frequency nanosecond pulse(≥10 kV/cm),the former has the advantages of no skin burned,no muscle contraction with pulse frequency.However,the application of low field and high frequency nsPEFs in clinical treatment needs to be optimized in parameters,and the problem of safety and mechanism need to be studied.Therefore,in this paper,the effect of low field and high frequency nsPEFs on the human melanoma cell line A375(A375 cells)was studied at the cellular level and animal level.The main results of this paper are as follows:(1)A nanosecond pulse generator for cell and animal experiments was establishedThe nanosecond pulse generatorwe used in this paper is under thetechnical support of Professor Mi Yan who is from the department of electrical engineering and high voltage and insulation technology of Chongqing University.In this case,we’ve successfully constructed the nsPEFs apparatus with 0-10 kV/cm field adjustable,frequency adjustable and adjustable pulse width pulse 100-500 ns.(2)Thermal effect evaluation of nanosecond pulsed electric fieldIn order to eliminate the effect of nsPEFs on the A375 cells lead to the lethal effect of cell death,this paper uses the infrared temperature sensor to detect the STM buffer solution temperature change under nsPEFs exposure.We through the measurement of field with 5 kV/cm,500 ns,100 pulses/string,500 pulse strings,string frequency 100 kHz and external fixed frequency 1 Hz nsPEFs,at this condition,the temperature of STM buffer solution increased 8.6 oC.From literature,we can calculate the nsPEFs,under this condition,whose output energy is 62.5kJ/m3.So that,we use this as the upper limited output energy of nsPEFs,and design the nsPEFs parameters with pulse width at 100,250 and 500 ns for the following experiments.Experimental parameters are shown in table 3.1.(3)Effects of nanosecond pulsed electric field on the biological behavior of A375 cells500 ns pulse width ns PEFs detected in all fields 5 kV/cm or 8 kV/cm and frequency under the condition of 100 kHz were mainly induced A375 cells necrosis.Under the pulse width of 250 ns nsPEFs,in the condition of high frequency more than 100 kHz induced A375 cells necrosis,under the condition of low frequency below 50 kHz nsPEFs inducing A375 cells necrosis and apoptosis both were no obvious effect.100 ns pulse width of nsPEFs detected in all field 5 and 8 kV/cm and the frequency under the condition of 1 MHz inducing A375 cells necrosis and apoptosis were not significantly affected,either.Further experiments showed that the field of 5 kV/cm or 8 kV/cm,100 ns,100 pulses/string,100 pulse strings,string frequency in 1 MHz and external fixed frequency 1 Hz,and 8 kV/cm,250 ns,100 pulse strings,100 or 200 pulses/string,string frequency 50 kHz and external fixed frequency 1 Hz,the all four condition of nsPEFs,which have significant inhibitory effects on A375 cells viability,migration and cell clone ability and cell cycle of biological behavior.The experimental results also showed that the combined treatment of nsPEFs with 50 nmol/mL DTIC on the effect of A375 cells viability,there is no synergistic effect on cell viability inhibition,and the combined treatment of nsPEFs with 50 μg/m L DDP on the effect of A375 cells viability,the inhibition on cell viability has a strong synergistic effect,and with the prolong of time,the synergistic effect may also enhanced.(4)Therapeutic effect ofnsPEFs on A375 cells transplanted subcutaneously in nude miceWe constructed animal model of A375 cells transplanting subcutaneously in nude mice,and then examined 5 kV/cm,250 ns,100 pulse/string,100 pulse strings,string frequency of 1 MHz and external fixed frequency 1 Hz nsPEFs on tumor of melanoma cells subcutaneous transplantation in nude mice treatment effect,the experimental results show that nsPEFs has effect on melanoma growth inhibitory.