Study Of The Anti-tumor Effect On Uterine Cervix Caicinoma By NDV HN Gene And CAV Apoptin Gene In Vitro And In Vivo

Cancer constitutes one of the most important medico-biological problems remaining generally unsolved at the beginning of the new century. Although involvement of numerous genetic factors in the pathogenesis of neoplasia is well proven, understanding of the complex molecular mechanisms underlying neoplastic growth is still disappointingly incomplete. Intense investigation of cancer-related genetic alterations at the somatic cell level has shed some light on the sequence of molecular events leading to malignant transformation of somatic cells and clonal neoplastic growth. It is now believed that most (and probably all) types of cancer share a relatively small number of molecular, biochemical and cellular traits called acquired capabilities. As that following six alterations relevant for malignancy: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Indeed, neoplastic growth should not be regarded as a cell-autonomous process intrinsic to the cancer cell. There is no doubt that cancer development strongly depends upon changes in interactions between malignant cells and their normal neighbours. For many cancers, conventional therapies render patients free of disease but relapse and death remains high, particularly in advanced cancers. Thus, there is great interesting developing novel therapies that can completely remove residual disease and prolong life. Although many novel approaches have been advanced in recent years, interest in cancer gene therapy has been exceptionally strong. The development of genetically engineer viruses and viral proteins that selectively target various tumors resulting in minimal toxicity in the normal tissues has emerged as a potentially important approach for cancer gene therapy.To investigate the mechanisms of effect on human cervical carcinoma cells Hela with Hemagglutinin-neuraminidase from Newcastle disease virus and Apoptin from from chicken anemia virus. We constructed recombinant plasmid pSFVHN and pSFVApoptin. The above recombinant expression plasmid was identified by enzyme and transfected into human cervical carcinoma Hela cells, then the recombinant was screened by using RT-PCR, western-blot and indirect immunofluorescence. The results showed that the apoptin gene and HN gene transcribed and expressed correctly in Hela cells. Hela cells were transfected with pSFVHN and pSFVApoptin and the anti-tumor effects in vitro were observed by using microscope. The results showed that the recombinants has evident lethal effects on Hela cells and the effects showed non-limit time-dose dependent relationship. The lethal effects were reinforced with the growth of transfection time and the concentration of the recombinant. The peak was reached 48 hours after infection when the concentration of the recombinant was 20μg/ml, the value is 59.3%和51.5%. AO/EB staining and DAPI staining were used to observe the cell morphological diversity, the results showed that pSFVHN and pSFVApoptin lead to the cell pyknosis, chromatin margination and apoptosis finally. DCFA staining to detect the Reactive oxygen species level, Rhodamine123 staining to detect the mitochondrial trans-memebrane potential and anti-MHC-I Mab coherent to detect the MHC-I level were used to observe the mechanism of anti-tumor effect of pSFVHN and pSFVApoptin and meanings of the pSFVHN and pSFVApoptin in activating immune. The results showed that the recombinant caused the decline of mitochondrial trans-membrane potential and step up of Reactive oxygen species level. In conclusion, the transfection of pSFVHN and pSFVApoptin ultimately induced Hela cell apoptosis and the apoptosis induce by the recombination is mainly through mitochondrial pathway.To observe the therapeutic alliance effects of apoptin gene and HN gene, Hela cells were infected by recombinant fowlpox virus vFVApoptin and vFVHN. RT-PCR, Western blot and indirect immunofluorescence was used to identify the expression of vFVApoptin and vFVHN. The results showed that the recombinant transcribed and expressed foreign protein magnanimously. MTT assay was used to detect the anti-tumor effects of vFVApoptin and vFVHN on Hela cells. The results showed that the recombinant can kill the tumor cells effectively as described below: 65.9%和55.8%. AO/EB and DAPI staining was used to observe the cell morphological diversity, the results showed that the recombinant fowlpox viruses lead to the cell pyknosis, chromatin margination and apoptosis finally. DCFA staining to detect the Reactive oxygen species level, Rhodamine123 staining to detect the mitochondrial trans-memebrane potential were used to observe the mechanism of anti-tumor effect of vFVApoptin and vFVHN. The results showed that the recombinant caused the decline of mitochondrial trans-membrane potential and step up of Reactive oxygen species level. In conclusion, the transfection of vFVApoptin and vFVHN ultimately induced Hela cell apoptosis and the apoptosis induce by the recombination is mainly through mitochondrial pathway. On the other hand, we applied FCM to detect the level of MHC-I, The results showed that the recombinant fowlpox viruses lead to up-regulation of MHC-I molecule.Furthermore, C57BL/6 mice model bearing H22 hepatoma was constructed by transplanting H22 cells into the right hind limb of the mice and the combined or single anti-tumor effect on H22 hepatoma of HN gene and apoptin gene was observed through the detection of anti-tumor ratio, survival rate, categorization of subset of splenic lymphocyte, CTL activity and the immuneostimulation. The results showed that that the growth of established tumors in mice immunized with the recombinants was significantly inhibited and the survival rate was prolonged. Furthermore, the immunization of mice with the recombinants elicited strong tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, CD4~+,CD8~+ T cells from mice vaccinated with the recombinants were elevated. Indicating that vaccination with the recombinants may be a novel and powerful strategy for cancer bio-therapy and the precise mechanism is on going investigation.