Research Of The Antitumor Effect On Laryngeal Carcinoma By Combined Application Of NDV HN Gene, CAV VP3 Gene And IL-18 Gene

Malignant tumor is one of the most severe diseases of human being currently. Theeffectively treatment or control of tumor is imminent because its disease incidence and casefatality have a tendency of ascensus year by year. With the rapid development of molecularbiology, genetic engineering and tumor immunology, cancer gene therapy has been seen asone of most possible means to cure tumor. Current methods of cancer gene therapy have someanti-tumor effects theoretically, however, the therapeutic effect in vivo is less obviously thanin vitro. Tumor progress is a multi-factor, multi-stage and multi-gene process, to cure cancer,we think that the combination apply anti-cancer gene is more effectively rather than singleapplication of anti-tumor gene. To study the strategy of cooperative cancer gene therapy, wecombined apply three anti-tumor gene, CAV VP3 gene, NDV HN gene and IL-18 gene, witheukaryotic expressive plasmid and fowlpox virus as vector to construct recombinant(pVVP3IL-18HN and vFVVP3IL-18HN) and design experiment to research the anti-tumoreffects of pVVP3IL-18HN and vFVVP3IL-18HN in vivo and in vitro. The study establish asolid foundation of combined cancer gene therapy, and has considerable significance indeveloping safe and/or effective anti-tumor medicine.The recombinant expression plasmid of pVVP3IL-18HN was constructed by insertingVP3 gene and promoter-phored(IRES) IL-18HN fused gene into the downstream ofpromoter(CMV). The above recombinant expression plasmid was identified by enzyme andtransfected into human laryngeal carcinoma Hep-2 cells, then the recombinant was screenedby using RT-PCR, western-blot and indirect immunofluorescence. The results showed that theVP3 gene and IL-18HN fused gene expressed correctly in Hep-2 cells and HN anchored oncytomembrane, however, apoptin concentrate in nucleus.Hep-2 cells were transfected with pVVP3IL-18HN and the anti-tumor effects in vitrowere observed by using microscope. The results showed that the recombinant has evidentlethal effects on Hep-2 cells and the effects showed limit time-dose dependent relationship.The lethal effects were reinforced with the growth of transfection time and the concentrationof the recombinant. The peak was reached 72 hours after infection when the concentration ofthe recombinant was 20μg/ml, the value is 61.9%.AO/EB staining and electronmicroscopedetection were used to observe the cell morphological diversity, the results showed thatpVVP3IL-18HN lead to the cell pyknosis, chromatin margination and apoptosis finally. DNAelectrophoresis, DCFA staining to detect the Reactive oxygen species level, Rhodamine123staining to detect the mitochondrial trans-memebrane potential and anti-MHC-I Mab coherentto detect the MHC-I level were used to observe the mechanism of anti-tumor effect ofpVVP3IL-18HN and meanings of the pVVP3IL-18HN in activating immune. The resultsshowed that the recombinant caused the decline of mitochondrial trans-membrane potential,fragmentation of genomic DNA and step up of Reactive oxygen species level. In conclusion,the transfection of pVVP3IL-18HN ultimately induced Hep-2 cell apoptosis and the apoptosisinduce by the recombination is mainly through mitochondrial pathway. Furthermore,C57BL/6 mice model bearing H22 hepatoma was constructed by transplanting H22 cells intothe right hind limb of the mice and the combined or single anti-tumor effect on H22 hepatomaof HN gene, apoptin gene and IL18 gene was observed through the detection of anti-tumorratio, categorization of subset of splenic lymphocyte, CTL activity and theimmuneostimulation. The results showed that the combinative application of HN gene,apoptin gene and IL-18 gene resulted in the significant anti-tumor effect andimmuneostimulation than HN gene, IL-18 gene or apoptin gene alone. In conclusion, apoptingene, IL-18 gene and HN gene in the same vector can result in synergistical anti-tumor effects.The precise mechanism is on going investigation.Since the fowl pox virus can infect the mammalian cells with a high infection rate andadoptive infection, the virus was studied as a hopeful expression vector. The recombinantexpression plasmid of pUTA2VP3IL-18HN was constructed by inserting VP3 gene andIL-18HN fused gene into the downstream of combined promoter(ATI-P7.5×20). Therecombinant expression plasmid and fowlpox virus 282E4 strain were co-transfected intochick embryo fibroblast cells, then the homologous recombination occurred. The recombinantfowlpox virus vFVVP3IL-18HN were screened by using BrdU and identified by RT-PCR,western blot and indirect immunofluorescence. The results of RT-PCR, western blot andindirect immunofluorescence showed that foreign gene carried by recombinant fowlpox viruscould express efficiently in tumor cells, and HN located in cell membrane, while apoptinlocated in the nucleus.To observe the therapeutic alliance effects of apoptin gene, HN gene and IL-18 gene,Hep-2 cells were infected by recombinant fowlpox virus vFVVP3, vFVHN andvFVVP3IL-18HN. Indirect immunofluorescence was used to identify the expression ofvFVVP3IL-18HN. The results showed that the recombinant expressed foreign proteinmagnanimously and HN located in cell membrane, while apoptin located in the nucleus. MTTassay was used to detect the anti-tumor effects of vFVVP3, vFVHN and vFVVP3IL-18HN onHep-2 cells. The results showed that the recombinant can kill the tumor cells effectively asdescribed below: vFVVP3IL-18HN>vFVVP3>vFVHN(vFVVP3IL-18HN vs vFVVP3,P<0.05;vFVVP3IL-18HN vs vFVHN, P<0.01). The results viewed that combined genetherapy is better than single application of anti-tumor gene. AO/EB staining was used toobserve the cell morphological diversity, the results showed that apoptin-containedrecombinant fowlpox virus lead to the cell pyknosis, chromatin margination and apoptosisfinally. Then we applied FCM to detect the level of MHC-I and CTL assay to analysis theimmuneostimulation of the recombinant. The results showed that HN-contained recombinantfowlpox virus lead to up-regulation of MHC-I molecule and specific CTL activity of PBMC.Moreover, the immuneostimulation effects of vFVVP3IL-18HN is more significant thanvFVHN(P<0.05), showed that HN gene and IL-18 gene may co-operate in educing anti-tumoreffects. DCFA staining to detect the Reactive oxygen species level and Rhodamine123staining to detect the mitochondrial trans-membrane potential were used to observe the deathmode of Hep-2 induced by recombination fowlpox virus infection and which pathway isinvolved in the cell death process. The results showed that recombination fowlpox virusinfection caused the decline or loss of mitochondrial trans-membrane potential and theinfection ultimately induced Hep-2 cell apoptosis and the apoptosis induce by recombinationfowlpox virus is mainly through mitochondrial pathway.To analysis the contribution of prime-boost strategy in this study, C57BL/6 mice modelbearing H22 hepatoma was constructed and the two recombinant (vFVVP3IL-18HN andpVVP3IL-18HN) described as above were used. With the detection of categorization of subsetof splenic lymphocyte, specific CTL activity and the tumor suppression ratio, the effect ofdifference inoculation strategy was analyzed. The results showed that the cellularimmunologic response and anti-tumor ratio of prime-boost group were much better than theother two groups. The experiment proved that the strategy of applying rDNA as Prime andrFPV as boost might lead to more effective anti-tumor immunity than other strategy, however,the strategy of applying rFPV as Prime and rDNA as boost showed almost the equivalenteffect of single application of rFPV or rDNA. In conclusion, suitable strategy hasconsiderable effect in cancer gene therapy and research.