Expermental Studies Of In Vivo And Viteo Pulsed Electric Field Combined With IL-21 Electrotransfer For Cervical Cancer Therapy

Cervical cancer is one of the commonest malignant tumor in female reproductive system.Now epidemiological investigation shows that the cases will develop in about 500 000 women this year worldwide. The primary cause in development of cervical cancer is human papillomavirus (HPV)infection,smoking and lower immunological function and so on.The patients in advanced stage cervical cancer and recurrence were immunological deficient and poor outcome.Other patients in earlier stage ,even though hysterectomy and local treatment may be recovery.But their quality of life hard to development, abortion and even reproductive function lose. So explore new treatment strategy is imminent.Biotherapy as a treatment modality of tumor has been extensively studied.To development outcome,survival quality and reproductive capability after effective surgerg, Immunotherapy has considerable potential for the treatment of cancer,cytokine therapy is one of the alternative. It can induction of tumour-specific cytotoxic T lymphocytes (CTLs) is thought to play a critical role in mediating the antitumour effects. Moreover, the ability of different cytokines to enhance NK-cell activity either alone or in the presence of tumour-specific antibodies. Cytokines, the biological response modifiers of the immune system, have had mixed success in cancer therapy. IFN-α,CSF,TNF-αand IL-2 are prototypical examples of successful cytokine-based immunotherapy for cancers. One such immune-stimulating substance is IL-21—a cytokine that belongs to the IL-2 superfamily of cytokines that all have a unique receptor but share a commonγc chain (γc) receptor—that has shown promising results in preclinical tumour models. we will discuss how the potential use of IL-21 in combination with other mothed may lead to better responses .The vectors used for gene transfection had a wide variety types,such as adenovirus,liposomes,etc.These vectors have their own deficienc,such as biological unsafety, higher immunogenicity and unstable,while the pulsed electric field as a vector for gene transfection can overcome these adverse factors. Electroporation is the permeabilization of the cell membrane with electrical pulses applied across the cell. electrical field induces instabilities in the polarized cell membrane lipid bilayer then alters its shape forming aqueous pathways that possibly are nano-scale pores named electroporation.It can be divided into RE and IRE.RE is commonly used to electrochemtherapy and Electrogene-therapy. IRE ,nevertheless, was used primarily for ablation of microorganisms and cells in vitro and of undesirable tissues in vivo. IRE should facilitate real time control of electroporation during clinical applications. There appears to be complete ablation to the margin of blood vessels without compromising the functionality of the blood vessels. Specific inductive capacity of tumor cell is larger than that of normal cell due to karyoplasmic ratio of tumor cell larger than that of normal one. so identical field strength effect,tumor cell is easier sfuffer to lesion.IRE is a promising method for treatment of tumors near blood vessels. Cervical cancer could also benefit from this property of IRE.With electric field gradually weaken,cells far away electrode will from IRE to beome RE. Tumor cells of RE within the tissue may be proliferation then tumor recurrence.So we will IRE combine with IL-21 electrotransfer for cervical cancer cell to therapy this disease, aim to IRE can ablate substantial volumes of tumor tissue and electrotransfect IL-21 to cells which were .reversible electroporated by pulse electric field. Tumor cells which were not ablated can transfection IL-21 gene for alter their immunogenicity and enforce immunity to tumor of patiens. CHARPTTER ONE: CERVICAL CANCER CELL BIOLOGY ALTERATION WITH ELECTROPORATION AT DIFFERETION ELECTRIC STRENGTHSOBJECTIVE: when in vivo tumor was treated by irreversible electroporation, no matter what shape of the electrode array, complete uniform pulsed electric field is difficult to achieve and there is always a gradient variation.In this study, cervical cancer HeLa cells were treated by pulsed electric field with different intensity, we aimed to explore the biological effects of changed electric field on tumor cells in vivo.METHODS: 9 groups of HeLa cell suspension with field strength from 0.5kV/cm to 2.5kV/cm(the gradient was 0.25kV/cm) and the control group were included. Pulse width was 100μs and frequency was 1Hz.Every 8 pulses was a set. Trypan blue was used for calculating cell mortality after the cells were treated for 0h, 6h and 24h;Giemsa staining of 6h creep plates was used to prove dose-effect relationship between electric field and cells. The expression of apoptotic protein caspase-3 was detected by SP immunohistochemisty; MTT was used for detection of cell viability 24 hours after treating;flow cytometry was used for detection of cell cycle change and apoptosis;RESULT: 0h trypan blue cell count showed that no significant difference was found between cell suspension with 0-1.5kV/cm field strength and there was significant difference between the control group and cell suspension with 1.75 - 2.5kV/cm field strength;6h and 24h cell count showed significant difference between the control group and cell suspension with 1-2.5kV/cm field strength,which was also showed directly by Giemsa staining;Caspase-3 expression after 6h creep plates was detected by immunohistochemisty and the results showed that positive signal in cell suspensions with 1.5-2.5kV/cm field strength was significantly higher than that of control group;flow cytometry showed significant difference of apoptosis between control group and cell suspension with 2-2.25kV/cm field strength,apoptosis in 0.5-1.75 kV/cm group showed increasing trend compared with control group;flow cytometry showed cells in G2 phase increased significantly when the field strength was 2-2.5kV/cm;24-hour MTT results showed no difference of proliferation between cell suspension with 0.5-1.75kV/cm field strength and cell proliferation was markedly inhibited when the field strength was 2-2.5kV/cm.The cells were ablated completely without heat superposition under the gap action of 3 sets of 1.75-2.5kV/cm field strength. The maximum of temperature rise of pulsed electric field is 4.9±1.17 under air convection and proper temperature;CONCLUSIONS: With the increase of electric field,cells developed from reversible electroporation to Irreversible electroporation, in this process field strength is as important as the number of pulse.IRE can be used alone for the treatment of cancer and its thermal effect can be ignored.Cell death may involved irreversible electroporation and cell apoptosis pathway.It is noteworthy that complete destruction of tumor cells should be achieved when pulsed electric field was used for tumor ablation,otherwise the change of cell cycle may lead to tumor recurrence and accelerated growth.CHARPTTER TWO: BIOLOGY EFFECTIVE TO HELA CELLS AFTER TRANSDUCTION IL-21 GENE FIRST ARTICLE :HOMO-IL-21 GENE CONSTRUCTION AND IDENTIFICATIONOBJECTIVE: IL-21,which Th1 cytokine is newly discovered possess extensive function of immunological regulation in antitumor. It is secreted by CD4+ T cell.To gain IL-21mRNA, gather mRNA was extracted by Trizol way from human peripheral blood lymphocyte that was costimulated with CD3/CD28 antibody.METHODS: Human peripheral blood lymphocyte,which was draw off by gradient centrifugation ,was co-cultured with CD3/CD28 antibody in vitro. Gather mRNA ,which was extracted by Trizol way from human peripheral blood lymphocyte, was amplificated under primer existence by RT-PCR and purified. IL-21cDNA and pcDNA4/HisMax vehicle were cutted and then conjuncted by XhoI/HindlII and T4DNA ligase respectively.pcDNA4/HisMax-IL-21 was transformated to competence TOP10 cells(CaCl2).Plasmid pcDNA4/HisMax-IL-21 was bulk extracted by Qiagen-tipl00 kit. Plasmid was identificated by XhoI/HindlII dizymase cutted, 1% agarose gel electrophoresis and sequencing.RESULT: The strap of amplification with RT-PCR was compared to Manke .It is show that length is 489bp and consistent with IL-21cDNA. There were two specificity straps of 5.3kb and 489bp respectively was appeared by dizymase cutted the plasmid. These are identical with fragment of pcDNA4/HisMaxA and IL-21 gene.Sequencing result show that gained IL-21 sequence is identical with that of genebank publication by BLAST programme to analyze in NCBICONCLUSIONS: Amplification of IL-21cDNA is succeed under primer effected on mRNA extracted after costimulated CD3/CD28 antibody and hPBL.SECOND ARTICLE:BIOLOGY EFFECTIVE TO HELA CELLS AFTER TRANSDUCTION IL-21 GENEOBJECTIVE: To search for biology effect of cervical cancer HeLa,which was transducted IL-21 by reversible electroporation.METHODS: We apply electricity parameter that was optimized to electroblot HeLa cells with homo-IL-21 plasmid by reversible electroporation. The plasmid has resistivity to Zeocin. We have progressed elective culture to HeLa cells on the basis of its peculiarity and gained HeLa–IL-21 and HeLa-vector cell lines.HeLa-IL-21 cells was compared cell viability and with HeLa and HeLa-vector cell lines by growth curve, MTT. Flow cytometry was used comparison their apoptosis and cell cycle.RESULT: Comparison viability of HeLa-IL-21 cells with HeLa and HeLa-vector cells showed not significant difference respectively. Their cycle and apoptosis showed yet not significant difference respectivelyCONCLUSIONS: HeLa,which was transducted IL-21 has not produced biology peculiarity alteration CHARPTTER THREE: TRANSDUCTION OF THE IL-21 GENES IN HUMAN CERVICAL CANCER CELLS PRODUCES T CELL-DEPENDENT AND IN-DEPENDENT ANTITUMOR EFFECTSOBJECTIVE: To examined whether novel cytokines, interleukin (IL)-21 that were expressed in HeLa cells could produce antitumor effects in the inoculated mice.METHODS: Human cervical cancer HeLa cells were electric transduced with human IL-21 and were injected into severe combined immunodeficiency (SCID) mice that were immune reconstituted by human peripheral blood lymphocytes (hPBL) and not reconstituted.Treatment of some of these SCID mices with anti-asialo GM1 antibody to depleted NK cells temporally in order to examine an involvement of T cells in the antitumor effects; We examined the production of IFN-γand Cytotoxic activities of spleen from SCID mice bearing parent and HeLa-IL-21 cells cells to HeLa and Caski.RESULT: Growth of HeLa-IL-21 tumors developed in SCID mice was completely inhibited compared with that of parent tumors, no matter whether in immune reconstituted or not immune reconstituted group. Growth of HeLa tumors developed in SCID with hPBL was retarded compared with in that of haven't hPBL. Afeter depleted NK cells temporally abrogated the growth retardation of HeLa-IL-21,the phenomenon in immune reconstituted group(IRG) were more significant than in nIRG. Cytotoxic activities of spleen cells to HeLa from spleen cells of SCID mice bearing HeLa-IL-21 cells with hPBL group was higher compare with in that of haven't hPBL and bearing HeLa group,the production of IFN-γof splenocyts from IRG to target cells were higher than that from nIRG and bearing HeLa group.CONCLUSIONS: Expression of IL-21 in HeLa cells can produce T cell dependent and -independent antitumor effects in an NK cell-defective condition.CHARPTTER FOUR: IRE COMBINED IL-21 GENE THERAPY CERVICAL CANCEROBJECTIVE: IRE can change to RE arounding tissues which electric field extinction is developed owing to its far away electrode. IL-21 may be transfected to relic tumor cells by RE then it produce antitumor effective.To explore effective and mechanism of IRE combination with RE as well as different electrode be used in treatment tumor. METHODS: The experiment parameter is composed of field,strength 2500V/cm,pulse width100μs and 1Hz frequency and every 8 pulses was a set . Bearing tumor naked mice and SCID mice were treated by pliers shape parallel and needle-electrode respectively. Bearing tumor SCID mice were divided to two groups of pulse and pulse addition IL-21 gene treatment, control group as well. We observe to their tumor regression and survival time. Histopathology was observed by HE staining after IRE treatrment 5 minutes,24 and 72 hours respectively.RESULT: No matter used pliers shape parallel or needle-electrode,they can not active suppression tumor growth to put 16 pulses into practice.In needle-electrode,however tumor accelerate growth after 16 pulses treatment short term. With increase number of pulses, IRE induce significant tumor regression especially in 64-80 pulses There are better results use of pliers shape parallel electrode than that of needle-electrode. Complete tumor regression occure in bearing tumor naked mice that were treated by 64 pulses There are not significant morphology alteration after 5 minutes with IRE treatment in tumour cell. However, the congestion of blood in the tumour vessels was evident. At 24 h, almost the entire tumor presented that the nuclei, extremely pycnotic, membranolysis.''cytoplasm''appeared to take delight in eosin staining. Under high power microscope,tumor cytomembrane were unintegrated, nuclei break into pieces and edge assemble. At 72 h, the nuclei were smaller even disappearance and tissue necrosis still more evident. Nevertheless,there are not morphology alteration of blood vessel and stroma cell to appear. There are significant different in tumor regression and survival time of bearing tumor SCID ,which were alone pulse treated and IRE combine with IL-21gene transfection At 12 day,tumor volume mean of IRE combine with IL-21 gene transfection and control group are 31.57±16.97 and 65.89±13.84 respectively.( p < 0.01) With increase number of pulses, Complete tumor regression in bearing tumor SCID mice of IRE combine with IL-21 gene transfection treatment was more evidentCONCLUSIONS:IRE can be used to effectively ablate tumors in vivo, Eelectric field used parallel electrode is uniformity over tumor area and effective to destroy tissue. Eelectric field reduction used needle-electrode is more evident and from IRE to RE. So,this property can transduce gene and impermeat anti-cancer to remaining tumor cell,which can supplementary to IRE treatment. IRE combine with IL-21 gene transfection is a effective tumor treatment mode...