Parameter Screening, Mechanisms And Animal Study Of Steep Pulsed Irreversible Electroporation Technology To Treat Lung Cancer

It has been revealed that steep pulse could kill tumor cells with its high-energy pulsed electric field to produce irreversible electroporation of tumor cells. Few are reported on the application of steep pulse with irreversible electroporation technology in lung cancer treatment. In the first part of our research, the effectiveness of the steep pulse irreversible electroporation technology was validated in human large cell lung cancer cells L9981. the optimized treatment parameters of steep pulse irreversible electroporation technology for large cell lung cancer cells L9981 was screened out. In the second part, the molecular effects of steep pulse were observed in the large cell lung cancer cells L9981 with the optimal treatment parameters. In the third part, the optimal treatment parameters of steep pulse irreversible electroporation technology were screened out for the nude mice lung transplant tumors L9981 in the nude mice subcutaneous transplant tumor model, and explored. To sum up, we have provided theoretical and experimental evidence for the possible application of steep pulse in the clinical treatment of lung cancer.Part 1 The parameters of steep pulse irreversible electroporation technology in human large cell lung cancer cell lines L9981Objective To validate the effectiveness of steep pulse irreversible electroporation technology in human large cell lung cancer cells, and to screen the optimal treatment of parameters for human large cell lung cancer cells. Methods Three different sets of steep pulse therapy parameters were applied on lung cancer cell L9981. The mortality of cell line L9981, cell inhibition rate and proliferation capacity of large cell lung cancer cell L9981 were detected through the Vi-Cell vitality analysis and by MTT. Results Steep pulsed irreversible electroporation technology for large cell lung cancer L9981 presents killing effects with various therapy *parameters. Conclusion Steep pulse irreversible electroporation technology for large cell lung cancer cell lines L9981 have better killing effects. The optimal treatment parameters are at voltage amplitude 2000V/cm, pulse width 100us, pulse frequency of 1Hz, pulse number 10 and repeated six times for lung cancer cells L9981. With this group of parameters, steep pulse could have the best tumor cell-killing cells effects.Part 2 Mechanisms for steep pulse irreversible electroporation technologyObjective To explore the mechanisms for steep pulse irreversible electroporation technology to kill the lung cancer cell L9981. Methods The apoptosis, cells mitochondrial membrane potential, internal PH changes and the intra-cellular calvium ions concentration were detected after steep pulses acted on the human large cell lung cancer cell L9981. Results Apoptosis test results indicated that cancer cells mainly experienced necrosis and apoptosis. Along with the increase of electric parameters, the proportion of the necrotic cells increased rapidly; The detection of cells mitochondrial membrane potential indicated that membrane potential occurred depolarization. Steep pulse can cause cancer cells to produce death and apoptosis.The PH value indicated that intracellular PH level down jumped. Internal PH became more acidic and led to cell death. The detection of intra-cellular calvium ions concentration showed that the number of free calcium significantly increased, and this change had killing effects on cell death and apoptosis.Conclusion Steep pulse could induce cell apoptosis.Part 3 Mechanisms of steep pulse irreversible electroporation technology in nude mice subcutaneous transplant tumor modelObjective Different steep pulse irreversible electroporation technology parameters have various effects on large cell lung cancer cells L9981 nude mice model,we try to explored the mechanisms for irreversible electroporation technology in vivo.Methods Three groups of steep pulse therapy parameters were applied to nude mice tumor model. With in vivo imaging system, we observed the effect of the different parameters. The pathological changes of tumor tissue and immune fluorescence detect Caspase-3 protein expression were also observed.Results With animals living imaging system, we found steep pulse had obvious inhibition effect for nude mice transplant tumor. Pathological findings showed that tumor tissue occured necrosis and apoptosis, Caspase-3 protein expression in tumor tissue increased compared with normal tissue.Conclusion Steep pulse irreversible electroporation technology has good inhibitory tumor effect for nude mice large cell lung cancer cells L9981 subcutaneous transplant tumor model. The best treatment parameters are with Splint type electrode, voltage amplitude 2000V/cm, pulse width 100us, pulse frequency of 1Hz, pulse number 60 and repeated three times for lung cancer cells L9981. Steep pulse induced coagulation necrosis of tumor tissue by inducing cell apoptosis.