Anti-hepatic Carcinoma Immunotherapy By Combined Application The Vaccines Of MAGE-1 And IL-18

PartⅠIntroductionCancer vaccine, which is tumor specific active immunotherapy, is a new tumor immunity therapy developed in 1990s. The fundamental principle is to separate and scavenge TSA and TAA, to prepare different vaccine and inject to tumor patients in vivo, then APCs uptake it and present it to immunocyte, sensitize and activate T-Lymphocytes, generate CTLs and specificly combine and kill tumor cells.Nucleic Acid Vaccines, also named genetic vaccine and DNA vaccine, is to clone gene fragment which is coding some kind of antigen to eukaryotic expression plasmid, then immune animal with the plasmid, the gene can express in the host and stimulate to creat humoral immunity and cellullar immunologic response. Genetic vaccine is composed of purpose gene and expression vector. Purpose gene is coding antigen cDNA sequence, it can be a individual gene or a battery genes which have synergism and protection function, and a length nucleotide sequence coding antigenic determinants, the gene fragment must be expressed in mammalian cell. Plamid is used as vector, because it can be produced with colibacillus and it can't be copied and integrated in mammalian cells. Antigen cDNA sequence is cloned in eukaryotic expression plasmid, then genetic vaccine is composed.Hepatic carcinoma is one of the most frequent malignant tumor in China and some Asian nations; it has high relapse rate and bad prognosis, and seriously threatens human's heathy and life. Now, we have progressed in the therapy of liver cancer, but it is not satisfied with the curative effect and prognosis. So, it is necessary to seek a better therapeutic tool. We'll do our best to provide a new method for therapy of liver cancer by tumor vaccine.MAGE-1 is one of the family members of MAGE, it does not express in normal tissues except testis and placent, but expresses in melanoma and many kinds of malignant tumors, and highly expressed in liver cancer. CTL can specificly recognize and kill tumor cells by recognizing expression product of MAGE-1. MAGE-1 antigen is suitable target of antigenic specificity anticancer therapy. IL-18 is a cytokine, which has much biologic activity, can promote proliferation and activation of NK cells, and induce NK cells to secrete IFN-γ. It can promote the express of MHCⅠby induction IFN-γand GM-CSF, and can strengthen immune effect of vaccine . In the research, we respectively construct recombinant plasmids of MAGE-1 and IL-18 to investigate if united application vaccine can improve anti-cancer effect.PartⅡThe construction and identification of recombinant plasmids of hIL-18 and MAGE-1Objective To construct recombinant plasmids of IL-18 and MAGE-1 respectively, and obtain correct recombinant plasmids as pcDNA3-hIL-18 and pcDNA3- MAGE-1, and validate bioactivity of them.Methods Scavenge RNA from liver cancer tissue and reverse transcript to cDNA, obtain hIL-18 fragment by PCR and recombinant plasmid of hIL-18 by T-A cloning, then it was enzymed by EcoR I and ApaⅠ, recovery enzymed fragment and link it to pcDNA3 fragment to construct pcDNA3-hIL-18.So did pcDNA3-MAGE-1.They were identified by PCR, enzymed and sequencing.Bioactivity of pcDNA3-hIL-18 was validated by RT-PCR and Western blot.Results Get 582bp fragment as hIL-18 and about 1000bp as MAGE-1 by electrophoresis. pcDNA3-hIL-18 and pcDNA3-MAGE-1 were identified correct by PCR, enzymed and sequencing. pcDNA3-hIL-18, pcDNA3-hIL-18 can express in transfected cell.Conclusion The recombinant plasmids pcDNA3-hIL-18 and pcDNA3-MAGE-1 which expressed IL-18 and MAGE-1 were constructed successfully, pcDNA3-hIL-18 had bioactivity after validated.PartⅢExperiment study of anti-cancer effect of combined vaccine as MAGE-1 gene and IL-18 factor in vitroObjective To detect lethal effect of genetic vaccines to MAGE(+)and MAGE(-) tumor cells.Methods Genetic vaccines were immunized to mice, spleen cell as effector cell, and SMMC-7721,BEL-7402,QQY-7701 hepatoma carcinoma cells as target cell, lethal effect of spleen cell to MAGE(+)and MAGE(-) tumor cells were validated. Kill ratio of different dose pcIL-18+pcMAGE-1 were detected for the best immune dose.Results Genetic vaccine groups had apparente lethal effect to MAGE(+)tumor cells, and they had significant deviation to control groups (P<0.01). Kill ratio obviously heightened(P<0.05), lethal effect of pcIL-18+pcMAGE-1 group was obviously higher than other groups in the same ratio , it was statistically significant to pcMAGE-1 group(P<0.05).Kill ratio of pcIL-18+pcMAGE-1 50μg+100μg group was highest and was statistically significant to pcMAGE-1 and other groups(P<0.01). Meanwhile, genetic vaccine groups had determinate lethal effect to MAGE(-)tumor cells, and they had statistically significant to control groups (P<0.05).Conclusions Lethal effect of pcIL-18+pcMAGE-1 group was highest to MAGE(+)tumor cells, combined application of IL-18 and MAGE-1 can further reinforce immuno-competence of vaccine.PartⅣConstruction of tumor-bearing mice model for MAGE(+)liver cancerObjective To get Hepa1-6 cell line which stably express MAGE-1 gene, to construct MAGE(+) tumor-bearing mice model, and to observe reproductive activity and tumorigenesis activity of transfected cell.Methods pcMAGE-1 which had been pured was transfected to Hepa1-6 cell, be validated by PCR and Western blot, then Hepa1-6-MAGE-1 cells were obtain. Different densities of Hepa1-6-MAGE-1 cell were inoculated to C57BL /6 j mice to research suit density for gene vaccine. Cell Growth and proliferation of Hepa1-6 and Hepa1-6-MAGE-1 were detected by MTT, they were inoculated to right-sidedness back subcutaneous of C57BL /6j mice respectively and tumor diameter was measured to observe tumorigenesis activity of transfected cell.Results mRNA and protein express of MAGE-1were detected in Hepa1-6-MAGE-1 cells by PCR and Western blot, the suit density of Hepa1-6-MAGE-1 cells for gene vaccine was 2×106, it had no statistica difference in cell proliferation of two groups. The mice were all infected with tumor in two groups, and there was no discrepancy in tumor size. Conclusion: The Hepa1-6 cell line which can stably express human MAGE-1 gene is established with good proliferation and tumorigenesis ability.PartⅤExperiment study on anti-cancer effect of combined vaccine as MAGE-1 and IL-18 in vivoObjective To research immune function influence and security of vaccine to mice, and to observe tumor growth and survival time effect of different immune function to tumor-bearing mice. Methods Spleen T cell subgroup of immuned mice was detected by FCM.Content of IFN-γ,IL-4,GM-CSF in pleen cell supernatant of immunized mice were detected by ELISA. Every mouse of experiment groups was injected Hepa1-6-MAGE-1 cells 2×106. Genetic vaccines were injected to mice, two booster injection were carried out at ten days intervals. Tumor growth and survival rates of vaccine immunized MAGE(+) and MAGE(-)mice were recorded.Results pcmIL-18+pcMAGE-1 group had statistic difference with pcmIL-18,pcMAGE-1 groups in CD3+,CD4+,CD4+/CD8+(p<0.05), pcmIL-18+pcMAGE-1 group significantly heightened(p<0.01). IFN-γ, IL-4, GM-CSF of pcmIL-18+pcMAGE-1 group was statisticly different with pcmIL-18,pcMAGE-1 groups( p < 0.05). IL-4 was not found in every group.Genetic vaccines can delay tumor growth time and lengthen live time of mice, pcmIL-18+pcMAGE-1 was better than the others. pcmIL-18+pcMAGE-1 was poor to inhibit MAGE(-) tumor. It was no pathologic lesion in liver,brain,kidney,lung of immunized mice,the vaccines had relative security.Conclusion Sym-injection vaccine of MAGE-1 and IL-18 had apparente anti-cancer tumor immunity.