The Mechanism And Effectiveness And Security Of Picosecond Pulsed Electric Field On Cervical Cancer Xenograft

Cervical cancer is one of common tumor of female reproductivesystem. Cervical cancer has been threat to women’s physical and mentalhealth seriously. The present treatment, to some extent, would damagepatients’ fertility and normal tissue. In recent years, cervical cancerpatients are getting younger gradually. And young patients are more urgentto keep reproductive function. Therefore, doctor and patients want to findmore treatments which can inhibit or kill tumor and reduce the trauma atthe same time.In recent years, many scholars concentrate to Pulsed Electric field(PEF) used in the biomedical field. Previous work showed thatmicroseconds (ms) and nanosecond (ns) pulse electric field havebiological effects on tumor cells, which can be coordinated chemotherapydrugs to kill tumor cells or kill tumor cells directly. These results lay afoundation for psPEF treatment of tumor. However microseconds andnanosecond pulse electric field must be on the carrier of the needle electrode to lead to tumor tissues, to some extent, which limitsapplication of the pulse electric field in clinical. If the pulse electric fieldcan be noninvasive lead to the body, then it can be applied to clinicalextensively. Therefore, picosecond pulse electric field provided a new wayfor the treatment of tumors, which is penetrating. However, there is lessresearch about psPEF treatment of tumor at home and abroad. Accordingto the "window effect" of pulse electric field and the electroporationeffects of microsecond pulse and the intracellular effects of nanosecondpulse. Theoretically, picosecond pulse electric field, which has the lowerpulse width, should have effect on organelles of the cell mainly. Andorganelles such as mitochondria and endoplasmic reticulum is related tocell apoptosis closely. Picosecond pulse electric field also has thefollowing advantages: high time resolution, high spatial resolution, andkeeping impulse characteristics of the input pulse in the process oftransmission and high resolution in the targeted area. Matching theimpulse radiating antenna, psPEF with excellent directivity and finehandling, can focus energy on the particular scope or make the targettissue necrosis. Radiation energy is concentrated both in space and in time.According to the feature, if psPEF could be transported into the tumortissue by IRA, the non-invasive treatment of tumors would come true.Previous study of our group identified that psPEF can induce hela cellapoptosis via mitochondrial membrane potential changes and may activating caspases through the endoplasmic reticulum stress pathwaypathway. However, these studies are limited to the cell research, and theactivities of the tumor cells in the body is affected by many factors. So themechanism, efficacy and safety of picosecond pulsed electric field on theanimal model was more significant for the further study of psPEF clinicalapplication.On the basis of the previous cell experiment, this study built thecervical cancer xenograft as tumor-burdened to research the mechanismand efficacy and safety of picosecond pulsed electric field on the animalmodel. The study would provide direct theory basis for the application ofpicosecond pulse electric field in clinical. The sudy is divided into threeparts.Chapter1The mechanism of inducing cell apoptosis of picosecondpulse electric field on cervical cancer xenograftPart1Building nude mouse xenograft of human cervical carcinomaObjective: To build nude mouse xenograft of human cervicalcarcinoma for animal experiments in vivo.Methods: We inoculated0.2ml Hela cell suspension per mouse tothe subcutaneous of back in nude mice, and could touch subcutaneousnodules after about1week. After about2weeks, we removed the tumortissue to transplant when the tumor diameter is about0.5cm-0.8cm. The diameter of transplantation tumor can reach1.0cm after about2weeks.The cervical cancer xenograft will be randomly divided into control groupand treatment group according to the design of following experiment.Results: The nude mouse xenograft of human cervical carcinomawere successfully established.Part2The study of appropriate parameters of picosecond pulsedelectric field on cervical cancer xenograftObjective: To study and select appropriate parameters of psPEF forcervical cancer xenograft.Methods: According to the results of previous experiment，we setparameters and design3treatment groups (electric field voltage:50kv,60kv and70kv), and fixed parameters as follows: pulse number:2000, pulsewidth:800ps, frequency:3Hz. The tumor of cervical cancer xenograftwas placed between tweezer-electrodes closely and non-invasively. Wekilled nude mice and removed tumor after24h. Then we used TTCstaining to measure and calculate the volume of necrotic tissue. Weselected parameter of psPEF for cervical cancer xenograft according to thestatistical results.Results: After psPEF treatment, it can be observed that pale areaincreased gradually with the increase of electric field voltage by TTCstaining. The results showed that the length and wide of necrotic tissue have no statistical difference between treatment groups. The thickness ofthe necrotic tissue have no statistical difference between50kV group and60kV group. The thickness of the necrotic tissue have difference between50kV group and70kV group(P<0.05). The volume of the necrotic tissuehave significant difference between three treatment groups (P<0.01).Conclusion: After psPEF treatment, as the voltage increasing thenecrosis tissue of cervical cancer increase significantly on cervical cancerxenograft. So appropriate parameters for the psPEF treatment on cervicalcancer xenograft were electric field voltage:70kv, pulse number:2000,pulse width:800ps, frequency:3Hz.Part3The mechanism of inducing apoptosis of psPEF on cervicalcancer xenograftObjective: To investigate the effect and the mechanism of inducingapoptosis of psPEF on cervical cancer xenograft.Method: We designed control group and treatment groups (6-h group,experiment12-h group and experiment24-h group which were executedand xenografts were taken out6h,12h and24h later after electric pulsesacted respectively), fixed parameters as follows: voltage:70kv, pulsenumber:2000, pulse width:800ps, frequency:3Hz. After anesthesia thetumor was placed between tweezer-electrodes closely and non-invasively.The histological changes were observed by HE staining and TEM. Cell apoptosis was determined with TUNEL assay. The mitochondrialtransmembrane potential was detected by LSCM, and the activity ofcaspase-3,-8,-9and-12was determined.Result: Pathological examinations did not demonstrate necrosis incontrol tumors, while the psPEF treatment led to cell necrosis to24-hgroup. The TEM demonstrated that the control tumors exhibited mildmitochondria swelling. In contrast, tumors with psPEF treatment showedearly coagulative necrosis in6-h group, notable coagulative necrosis andmany apoptosis bodies in12-h group and severe coagulative necrosisand few apoptosis bodies in24-h group. There was an increase of theapoptosis rate in treatment groups, compared with control (P<0.01). Theapoptosis rate of12-h group was significantly higher than other treatmentgroups (P<0.01). There was, however, no statistical difference between the6-h and the24-h group. Stained by the mitochondria-specific probes JC-1,the mitochondria of tumor can emit red and green fluorescence. Theexperiment6-h group and12-h group presented a reduced redfluorescence and increased green fluorescence when compared tountreated controls. However, The experiment24-h group showed red andgreen fluorescence weak than6-h group and12-h group. Caspase-3andcaspase-12activity were increased in tumors exposure to psPEF.Caspase-3and caspase-12activitiy in different groups followed thehierarchical pattern: the12-h group> the24-h group> the6-h group=the control group (P <0.05). Whereas caspase-8and caspase-9activity werenot affected by psPEF exposure.Conclusion: Our study demonstrated that psPEF can reach tumortissue of the cervical cancer animal models by tweezers-electrode and killtumor cells, in which apoptosis play an important role via the decreasedmitochondrial membrane potential and endoplasmic reticulum stress.Chapter2The effectiveness of psPEF treatment on cervical cancerxenograftObjective: To investigate the tumor size, relative tumor proliferationrate and survival time of cervical cancer xenograft after psPEF treatment.Methods: Cervical cancer xenograft were divided into control groupand treatment group. Parameters were set as follows: voltage:70kv, pulsenumber:2000, pulse width:800ps, frequency:3Hz. After anesthesia thetumor was placed between tweezer-electrodes closely and non-invasively.To observe the growth of tumor and the status of nude mice. The tumorvolume was measured one time every3days. Relative tumor proliferationrate was calculated after psPEF treatment. Cervical cancer xenograft ofcontrol group and treatment group were feed in the same condition andrecord the survival time and make survival curves.Results: Tumour burst in24days and58days in control group andtreatment group respectively. There were statistical difference after3days, 6days,9days between tumor volume of two groups. However, there wasno statistical difference after12days. Relative tumor proliferation ratewere76.87%,71%and68.87%in3days,6days,9days respectively.Survival time is (41-92) days and (70-118) in the control group andtreatment group respectively. There was no statistical difference insurvival time of mice between the control group and the psPEF treatmentgroup.Conclusion: To some extent, psPEF treatment had tumor suppressioneffect on cervical cancer xenograft, however, there was no obviouslong-dated inhibitory effect on tumor.Chapter3The security of psPEF treatment on cervical cancerxenograftPart1The security of psPEF treatment on the skin andsubcutaneous tissue around tumorObjective: To investigate psPEF treatment on the skin andsubcutaneous tissue around tumor on cervical cancer xenograft.Method: We designed control group and treatment groups (6-h group,experiment12-h group and experiment24-h group which were executedand the skin and subcutaneous tissue which psPEF conduct were taken out6h,12h and24h later after electric pulses acted respectively), fixedparameters as follows: voltage:70kv, pulse number:2000, pulse width: 800ps, frequency:3Hz. After anesthesia the tumor was placed betweentweezer-electrodes closely and non-invasively. The histological changeswere observed by HE staining and TEM. Cell apoptosis was determinedwith DNA ladder.Result: Pathological examinations did not demonstrate necrosis inthe skin and subcutaneous tissue of the control group and treatment group.The TEM demonstrated that epidermis and the dermis had no necrosis inthe control group and treatment group. Tumors with psPEF treatmentshowed that basal cell mitochondria was mild swelling and vascularendothelial cells was normal in6-h group, cell mitochondria was mildswelling in12-h group, much bubble fat cells was complete and vascularstructure was normal and endothelial cells was no swelling in24-h group.There was no DNA ladder in the control group and treatment group.Conclusion: There was no obvious cell apoptosis, injury and necrosisin the skin and subcutaneous tissue after psPEF treatment on cervicalcancer xenograft.Part2The effect of psPEF treatment on the important viscera ofcervical cancer xenograftObjective: To investigate the effect of psPEF treatment on the heart,liver, kidney of cervical cancer xenograft.Methods: Cervical cancer xenograft were divided into control group and treatment group. Parameters were set as follows: voltage:70kv, pulsenumber:2000, pulse width:800ps, frequency:3Hz. After anesthesia thetumor was placed between tweezer-electrodes closely and non-invasively.The blood, heart, liver and kidney were collected at24h after psPEFtreatment. The cardiac function, liver function and kidney function wereobserved by LDH Assay kit, ALT Assay kit, AST Assay kit and BUNAssay kit, respectively. The histological changes were observed by HEstaining.Results: There was no statistical difference in ALT, AST, LDH andBUN activity of cervical cancer xenograft blood between the controlgroup and treatment group. Pathological examinations did not demonstratenecrosis in the heart, liver and kidney of cervical cancer xenograft afterpsPEF treatment.Conclusion: There was no obvious function and histological changein the heart, liver and kidney after psPEF treatment on cervical cancerxenograft. Therefore, psPEF treatment have no effect on cervical cancernude xenograft.