Biological Behavior Change Of Residual Tumor After Irreversible Electroporation On Cervical Cancer Nude Mouse Model

Cervical cancer is the most common cancer of female reproductive system cancers, even in the whole body of the female malignant tumors,the incidence of cervical cancer is second only to breast cancer. Since cervical cancer is closely related to HPV infection and socioeconomic status, every year about 50 million new cases occur in developing countries,among which about 1/3 are happenning in China. In recent years, the incidence of cervical cancer was getting younger and younger, the incidence of cervical cancer in women under 37 years old increased significantly. Timely appropriate comprehensive treatment will get good outcomes for early cervical cancer patients, even get curred. However, for young patients of early cervical cancer, especially in patients above clinical stage Ⅰ, surgical treatment often causes serious damage to the reproductive organs(vagina, cervix and uterus), and affect sexual function and fertility. Therefore, looking for an effective treatment with the greatestpossiblity of protection of sexual function and reproductive capacity(fertility-sparing therapy) has been the common aspirations of both doctors and patients.Pulsed Electric Fields(PEF) is a newly tumor physical therapy. By adjusting the electric field pulse frequency(Frequency), the number of pulses(Pulses), pulse width, the electric field intensity/ voltage(Electric Field Strength/ Voltage) and other parameters, it can be generated different PEFs that play different biological effects. Wherein a kind of PEF with pulse width at μs level, and field strength of ~ k V/cm, can make the phospholipid molecules of the cell membrane rearrange, where the hydrophilic membrane channel forms and micropores can not be restored.It is a kind of permanent damage to the cell membrane. Briefly, cells are at high membrane permeability state after exposing to the electric field, and then an imbalance of biological macromolecules and ionic substances occur consequently both intracellular and extracellular, which is called irreversible electroporation(IRE). A machine called "nano-knife"(Nano Knife? System; Angio Dynamics, NY, USA) which based on this technology has been approved by the US FDA for clinical treatment of soft tissue tumors in October 2011.Our previous studies showed that the threshold of irreversible electroporation on He La cells in vitro 1750V/cm. Many researches have confirmed that IRE was an effective treatment for solid tumors. To date,IRE has been applied to solid tumors such as pancreatic cancer, liver cancer,kidney cancer, prostate cancer, melanoma and soft tissue tumors. It has been considered the most potential second-line alternative for unresectable tumors, or high surgical risk tumors.The effect of IRE dose not depend on thermal effects, which is different from Radiofrequency ablation laser therapy, yet there is still varying degrees temperature rising in the ablating region. Moreover, with the electric field attenuation, the surrounding residual tumor tissue will receive relatively low energy pulsed electric field, which may potentially affect the malignant behavior of tumor cells. Therefore, will there be a similar situation with RFA of promoting residual tumor proliferation, tumor recurrence and metastasis? If it dose promote proliferation, then how long is the appropriate time of follow-up and treatment interval? Are there opportunities for secondary ablation? These problems of IRE therapy have not been reported.Thence, the purpose of this study is by constructing Firefly Luciferase gene and green fluorescent protein gene double labled He La cell lines, then establishing residual tumor model, to quantitative analysis the changes of residual tumor biological behavior after IRE ablation. Meanwhile, noticing the RE region outside the IRE ablation, we plan to make full use of the RE region, to exploring the feasibility of combined threpy of IRE and gene therapy to treat cervical cancer nude mouse model. This topic is divided12 into the following three parts.PART ONE CONSTRUCTION AND SCREENING OF LUC / GFP DOUBLE LABELED HELA CELL LINESObjective: To construct and screen of LUC / GFP double labeled He La cell lines.Method: Connect PCR amplified LUC gene to the GV218 lentiviral vector, which contains the GFP reporter gene, to construct doubly labeled recombinant plasmid. Verified by sequencing, the correctly recombinant plasmid was transfected into 293 T cells, to packag and purify viral, then measure lentivirus titer by fluorescence assay. Transfect He La cells with LUC/GFP labeled lentivirus vectors, pressurized screening with puromycin to establish stable cell line according the resistance gene carrier. CCK-8method, propidium iodide staining and Western blot were used to detect cell cycle and whether transfection affecting biological behavior of He La cells.Results: Lentiviral vector was construced with lentivirus titer of 2.0 ×109TU/ml. After viral transfection and pressurized screening with puromycin, stable LUC/GFP labeled He La cell line was successfully constructed, and the cell biology has no change determined by CCK-8, PI staining and Western blot.Conclusion: The LUC/GFP labeled He La cell line constructed bytranstecting of lentiviral vector has no change in cell biology, which is suit for animal model study.PART TWO BIOLOGICAL BEHAVIOR CHANGE OF RESIDUAL TUMOR AFTER IRREVERSIBLE ELECTROPORATION ON CERVICAL CANCER NUDE MOUSE MODELObjective: To investigate the biological behavior change of residual tumor after irreversible electroporation on cervical cancer nude mouse model.Method: Xenograft model was constructed by tissue embedding method;the dose-effect relationship between the different field strength of IRE and the amount of residual tumor tissue was determined by in vivo imaging technology. Photoacoustic imaging was applied immediately after IRE to detect tissue blood supply of the residual tumor tissue; a control study among sham group, surgical resection group and IRE group was carried out and detected by in vivo imaging technology to quantitative analysis the fluorescence intensity. Specimens before treatment, 10 days post-treatment and 20 days post-treatment were harvested, then the changes of proliferation, invasion and metastasis related protein PCNA, VEGF and MMP-9 were detected by Western blot test.Results: A residual tumor[(53.22±6.28)%] model was established byIRE with parameters of 2000V/cm, 100μs, 1Hz, 15 pulses; Photoacoustic imaging confirmed there was tumor tissue left with blood supply.Quantitative analysis of the fluorescence intensity by in vivo imaging technology showed a decrease in surgical resection group and IRE group of(53.67±5.22)% and(51.81±4.39)%, respectively, while there was no significant change in shame group just like we expected. The fluorescence intensity increased significantly 10 days post treatment in surgical resection group compared with IRE group(P<0.05). While there was no significant change among the three groups 20 days post treatment(P > 0.05). Tumor related proteins detected by Western blot showed that PCNA expressed significantly lower in IRE group than the other two groups 10 days post treatment, while there were no remarkable changes in VEGF, E6 and MMP-9 among the three groups 10 days post treatment(P > 0.05), also there were no obviously difference of the above proteins among the three groups 20 days post treatment(P>0.05).Conclusion: A stable residual tumor model caused by IRE was established, IRE might inhibit the proliferation of residual tumor to some extent in a short period after performance, while has no obvious influence on invasion and metastasis.PART THREE FEASIBILITY OF COMBINED IRE AND GENE THERAPY TO TREAT CERVICAL CANCER NUDE MOUSE MODELObjective: To investigate feasibility of combined IRE and gene therapy to treat cervical cancer nude mouse model, and their coeffect on residul tumor.Method: Insert the HPV18 E6 gene sequence-specific interference structure into p Genesil-1 vect to construct HPV 18 E6 sh RNA eukaryotic expression vector. Cells were divided into 6 groups and subjected to corresponding treatments: Control; IRE; CTL sh RNA plasmid; IRE + CTL sh RNA; E6 sh RNA plasmid; IRE + E6 sh RNA. PCR, Western blot and CCK-8 were applied to detect the effect of combine treatment on He La cells. HE staining and frozen sections were used to detect the kill effect and GFP expression, respectively, 48 hours after the combined treatment on nude mice model. Sham group, IRE group, plasmid group and combined group were set up. The tumor volume were measured regularly to learn residual tumor growth curves, meanwhile, using Western blot method to detect the expression of tumor growth, invasion and metastasis related proteins.Results: HPV 18 E6 sh RNA eukaryotic expression vector was successfully constructed. The expression of GFP 48 hours after IRE indidicated IRE could mediate plasmid into the He La cells. The expression of E6 m RNA in combined treatment group decreased remarkablely in combined group compared with IRE group(P<0.05); Western blot showed E6 protein decreased(P<0.05), and P53 increased(P>0.05), while PCNA decreased(P < 0.05). CCK-8 indidicated the combination treatment of IRE and E6 sh RNA remarkablely inhibited He La cell proliferation versus control group(P < 0.05). HE staining and frozen sections confirmed the success of tumor tissue residue and the expression of GEP. Western blot showed combined treatment of IRE and plasmid could inhibit tumor growth, and had no significant effect on the invasion and metastasis.Conclusion: IRE can mediate plasmid into the He La cells both in vivo and in vitro; and the combined treatment can significantly inhibit the proliferation of He La cells, and inhibit the proliferation of residual tumor.IRE combining interference plasmid may have a potential of a new strategy for preventing residual tumor.