Research On The Combined Effects Of Pulse Electric Fields And Wild-Type P53 Gene On Apoptosis In Hela Cells

Cervical cancer is one of feminine reproductive system most common malignant tumors, the disease incidence rate is only inferior to breast cancer. Regarding the tumor treatment, therapies and so on surgery, chemotherapy, physics, have the varying degree at present , for this reason people have been seeking the more effective treatment strategy. In recent years, by its unique electricity perforation effect in the gene extension to perish and so on , pulse electric fields(PEFs) aspects to become the research weakly the hot spot.By its unique induction cell bioelectricity effects and so on electricity perforation as well as irreversibility electric breakdown, PEFs became a new way to studies and regulates the cell biological effect by the electrical scientists, the cell biologists and clinicians, It unfolded the good prospects for development and to the tumor complex therapy aspect obtained the preliminary application in clinical. Wild type p53 gene is the recognition damps the cancer, the p53 protein it codes in vivo by the tetramer form existence. When cell DNA is damaged, the p53 protein and the DNA corresponding spot union, causes the cell to stagnate in the Gl time,suppresses the unwindase activeness , And with the duplication factor A interaction, participates in DNA the duplication and the repair ,so that the cell can have the enough time repair to damage . If the repair defeat, induces the gene which through the upward perishes weakly, urges the cell to perish weakly, thus prevents the cell to become malignant .Therefore the p53 gene's function deactivation in HPV sends in cervical cancer process to play the strong character.This research is for the purpose of through in vitro experiment, transfection wild type p53 gene enters cervical cancer Hela cell through PEFs , Inspects the inhibitory action of Hela cell,then handle the cells with screened parameter PEFs ,observe the apoptosis.Part1 Wild type p53 transfected by PEFs and to Hela cell's influenceObjective transfect the wild type p53gene into Hela cells through PEFs , Obtains the Hela-p53 cells,which P53 protein stably expressed, observe grow influence of Hela cell induced by p53 gene.Method Uses the optimized PEFs transfect material particle ,which combined with combined with p53 gene and the spatial ,into Hela cells,through material particle Zeocin resistance screening obtaine the p53 gene and the spatial material particle HeLa cell (HeLa-p53and HeLa-vector),identified by RT-PCRand Wsetern bolt;Cell counting and MTT examines each group of cell's growth situation,plan cell growth curve; growth vigor of HeLa,HeLa-vector and HeLa-p53 cells by MTT.Results Obtains Hela-p53 cells which P53 protein stably expressed and the Hela-vector cell; Hela-p53 cell growth activeness is lower than other two groups of cells obviously.Conclusion Certain parameter combination's pulse electric field may transfer the material particle to enter the Hela cell; the wtp53 gene extension growth inhibitory action of Hela cells.Part2 The parameter screening of PEFs induce cervical cancer hela cell apoptosisObjective Observes a series of parameter combination the pulse electric field and induces to human cervical cancer Hela cells suppression and apoptosis; optimize the electric field parameter combination which induced the person cervical cancer Hela cell apoptosis. Detect the temperature rise of the cell fluid immediately after PEFs handled,to detect thermal reaction in the process of Hela cells apoptosis induced by PEFs.Method The choice electric field parameter for the frequency is 1Hz, the pulse width for 100μs, each time affects 8 pulses, the voltage respectively is 200, 300, 400, 500, 600, 700, 800 (V), supposes the control group, the altogether 8 groups, the PEFs handled, after 12h and 24h, the liver moss hopes the blue notation examination cell survival percentage; 24 hours later MTT examination cell vigor;After flowing the type cell technique to examine the cell apoptosis after 12h; thermo-couple survey the fluid temperature rise.Results The liver moss hoped that the typan blue counting cell survival percentage, compares the 200-400v group not obvious difference with the control group (P> 0.05), 500-800v group difference remarkable (P<0.05);the 500th, 600V group with 700, 800v group comparison difference has the significance (P<0.05);The MTT examination cell vigor result is similar to the cell counting result. the 800v group actual temperature rise is 4.5℃.Conclusion PEFs has the accurate inhibitory action to the cell, is containing necrosis and apoptosis; PEFs induction cell apoptosis has a electric field parameter area; In the process of PEFs suppressor cell, the thermal reaction function is not obvious.Part3 Research on the combined effects of pulse electric fields and wild-type p53 gene on apoptosis in Hela cellsObjective By the second part of screening pulse electric field parameter combination, processes Hela, Hela-vector, the Hela-p53 cell separately, then observation apoptosis of each group cells.Method Flow Cytometer to examine each group of cells apoptosis; western bolt examines apoptosis related protein Bax, Bcl-2 in each group of cell's expression; Confocal Laser Scanning Microscope(LSCM) to examine the casepase-3 protein in each group of cells expressionResults Flow Cytometer discovered that Hela-p53 group cell apoptosis rate is bigger than another two groups of cells obviously; Western bolt discovers Hela-p53 cells to press to apoptosis Protein Bax express obviously higher than Hela and Hela-vector cells, suppresses protein Bcl-2 to be opposite;apoptosis related protein Casepase-3 to be higher than another two groups obviously weakly in Hela-p53 cell's expressionConclusion the p53 gene may strengthen the effect of PEFs to induce human cervical cancer Hela cell apoptosis.