The Biology Behavior Of Irreversible Electroporation On Cervical Cancer Cells

Cervical cancer is one of the three most common malignant tumor in female reproductive system. In recent years, its incidence appeared new characteristics:the younger. At present cervical cancer treatments including surgery, chemotherapy and radiation therapy, and most patients can get a good treatment effect, but the structure and function of the reproductive system can be damaged after treatment. However, young patients have a strong desire to retain their fertility.Therefore, the research hotspot is find a cure, not only can be effective in the treatment of tumor, but also retain reproductive and sexual dysfunction.Pulse electric field is a new method of physical ablation tumor.Cells produce different biological effects processed the different pulse width of electric field,from reversibility electroporation, irreversibility electroporation to intracellular electromanipulation.Irreversible electroporation is one of the most widely research way of pulse electric field in recent years,and the main mechanism of its kill the tumor cells is the breakdown of the cell membrane irreversibly. At present a large number of studies have confirmed the validity of the irreversible electroporation kill tumor.And irreversible electroporation has the following advantages:the treatment time is short, curative effect does not depend on the thermal effect, the range of treatment is precision controlled according to the disposal of the needle electrode and the selection of pulse parameters, etc.Therefore, the irreversible electroporation show a broad application prospect in the field of tumor treatment.Tumor tissue is no clear boundary with the surrounding normal tissue due to its invasion, which makes treatment scope is not controllable, the edge of the pulsed electric fields in the process of ablation area will residual tumor cells inevitably. The biological behavior of residual tumor cells is a key to the prognosis of disease, and has the vital significance for the IRE used for clinical treatment safely.The research of the biological behavior of residual tumor cells after treated by IRE is very little.Therefore, in this study,we exposed HeLa and Siha cells to sublethal dose PEF, and detected the change of the biological behavior of residual tumor cell after the pulsed electric field processing, including proliferation, migration, invasion, adhesive ability and sensitivity of cisplatin, and discussed molecular mechanisms.Chapter 1 The effect on cells malignant biological behavior of Hela and Siha cells induced by IREPart 1 The study of appropriate sublethal parameters of irreversible electroporation on cervical cancer cellsObjective:To select appropriate sublethal parameters of IRE on cervicel cancer cells.Method:According to the results of previous experiment, we set parameters and design 3 treatment groups (number of pulse:8,16 and 24), and fixed parameters as follows:voltage:400V, pulse width:100us, frequency:1 Hz.The distance between the two electrodes of electric shock cup for 4mm.According to the formula field strength= voltage/distance, the field strength of this experiment is 1000V/cm. The Hela and Siha cell suspension be added to electric shock cup, and exposed to pulsed electric field of the different parameters respectively, but control group not be treated by pulsed electric field. We used cck-8 method to measure cell activity and calculate the survival rate after treatment.we select parameter of IRE for nearly half inhibiting cervical cancer cells according to the statistical results.Results:After IRE treatment, it can be observed that cell activity decreased gradually with the increase of number of pulse by cck-8 assay. The cell viability of Hela and Siha cells have significant difference between the three groups (P<0.05),and the cell viability of 16 pulses group was the closest to 50%.Conclusion:We chosen sublethal dose parameters of IRE ablating cervical cancer cells is field strength 1000V/cm, width 100us, the frequency of 1Hz, pulse number 16.Part 2 The effect of sublethal doses IRE on malignant biological behavior of cervical cancer cellsObjective:To observe the effects of sublethal doses irreversible electroporation on malignant behavior of cancer cells.Method:Hela and Siha cells were divided into control and treatment groups. The treatment group were exposed to Pulsed electric field of fixed parameters (field strength 1000V/cm, width 100us, the frequency of 1Hz, pulse number 16). The Cck-8 assay was used to detect cell activity and calculate cell survival rate.Cell cycle was determined with Flow cytometry. The cell proliferation situation was observed by CFSE fluorescence-labeled.The migration ability was measured by cell scratch assay and transwell cave.The invasion and adhesion ability were determined by transwell invasion experiment and matrix adhesion test.Results:The result of cck-8 assay was showed that Hela and Siha cells growth were decreased after treated by pulsed electric field in 24h.The cell cycle was arrested in phase G1. Hela and Siha cells in G1 phase was increased significantly (P<0.05) and S phase cells was decreased significantly(P<0.05). Cell proliferation ability significantly suppressed after pulsed electric field processing. The 5-6 generation cells were Significantly less than the control group(P<0.05).After treated by pulsed electric field,the ability of migration,invasion and adhesion in Hela and Siha cells had no Significantly influence. The result between the treatment and control group was no statistically significant difference(P<0.05).Conclusion:Lethel dose IRE can inhibit the proliferation ability of Hela and Siha cells. Cell migration, invasion and adhesive ability is not significantly affected.Part 3 The mechanism of inhibiting proliferation of sublethal doses IRE on cervical cancer cellsObjective:To investigate The mechanism of inhibiting proliferation of sublethal doses IRE on cervical cancer cells.Method:After the pulse treating at 24h, cells were collected and extracted proteins.We used Western Blot to detect the expression levels of cell proliferation related genes P53, P21, CDK2 and PCNA.Results:The expression level of P53, P21 in Hela and Siha cells increase, and PCNA, CDK2 decrease compared with the control group (P <0.05).Conclusion:The mechanism of inhibiting proliferation of sublethal doses IRE on cervical cancer cells is promote expression of P53-P21-CDK2 signaling pathway and decrease expression of PCNA.Chapter 2 The effect on cells cisplatin sensitivity of Hela and Siha cells induced by sublethal doses of IREObjective:To study The effect on cells cisplatin sensitivity of Hela and Siha cells induced by sublethal doses of IRE, and its mechanisms.Method:Cells of Pulse group and the control group were exposed to cisplatin solution in different concentration(5ug/ml、10μg/ml、15ug/ml and Oug/ml)for 24h. Cck-8 assay was used to detect cell activity and calculate inhibition rate of cisplatin. Cells treated with cisplatin solution was collected and broken.The cisplatin concentration of intracellular fluid measured by high performance liquid chromatography (HPLC). The expression level of copper transporter 1(CTR1) was detected by Western Blot assay.Results:The result of cck-8 assay showed that Cisplatin inhibition rate of cells was significantly increased after treated by IRE(P<0.05). The cisplatin concentration of intracellular fluid increased conpared to control group(P<0.05).After treated by IRE,the expression level of CTR1 was Significantly increased(P<0.05).Conclusion:Sublethal doses of IRE can increase the sensitivity of cisplatin in cervical cancel cells, and its mechanism is the amount of cisplatin accessing in cell increase by raising the expression of CTR1.