The Inhibitory Effect Of Embryonic Stem Cell On Mice Hepatoma In Vitro And Vivo

Section I The investigation of anti-proliferation effect of embryonic stem cell on mice hepatoma cell line in vitro.Objective: To study the anti-proliferation effect of embryonic stem cell on mice live cancer line hepa1-6 and related molecular mechanism.Methods: Coculture embryonic stem cell and hepa1-6 with Transwell chamber,culture hepa1-6 cells with embryonic stem cell conditioned medium,observed cancer cell growth with MTT methods.Study cell cycle and apoptosis of cancer cell with flowcytometry.Study cell cycle G1 phase related protein change with west-blotting.Results: Coculture embryonic stem cell and cancer cell or culture cancer cell with embryonic stem cell conditioned medium inhibited cell proliferation at different time point.Embryonic stem cell conditioned medium cultured cancer cell increased G1 phase cell cycle arrested and decreased G2/M phase cell cycle.Embryonic stem cell conditioned medium cultured cancer cell didn't increase cell apoptosis.West-blotting showed cultured cancer cell decreased cyclin D1-CDK4/ CDK 6Conclusions: Embryonic stem cell in vitro inhibited hepa1-6 cell proliferation not through induced cells apoptosis,but by arresting cells in G1 phase of the cell cycle,due to decrease expression of the cell cycle related proteins-cyclin D1-CDK4/ CDK 6.Section II Identification of microRNAs involved in growth arrest in embryonic stem cell microenvironment cultured hepa1-6 cellsObjective: To study microRNAs related to embryonic stem cell conditioned medium inhibited liver cancer cell proliferation and bio-information.Methods: miRNAs array analysis identified different expressed miRNAs with Agilent Mouse mi RNA chips and RT-PCR confirmed the results.Using microRNA database to predicate target genes of the deregulated miRNAs.Gene ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway related bio-informatics analysis.Results: MicroRNAs array analysis and RT-PCR identified 6 differentially expressed miRNAs(3 were upregulated and 3 were downregulated)and predicted 423 putative target genes of the deregulated miRNAs.Gene ontology(GO)analysis revealed that the putative target genes were associated with cell DNA synthesis,apoptosis and signal transduction etc.The bio-informatics analysis indicated that deregulated miRNAs involved pathway in cancer signaling transduction,wnt signaling pathway and hippo signaling pathway etc.Conclusion: Identified miRNAs might be though target cell cycle related m RNAs and wnt signaling pathway to regulate embryonic stem cell conditioned medium inhibited hepa1-6 cell proliferation effect.Section III Antitumor effect of embryonic stem cell vaccine against hepatoma in miceObjective: To investigate the inhibitory effect of embryonic stem cell vaccine against mice hepatoma at different tumor phase with different vaccine injection approach.Methods: At low tumor burden stage model,three groups of mice were inoculated with embryonic stem cell vaccine or PBS and tumor cell at same time,in vaccine groups inoculated with vaccine in tumor or subcutaneous.The tumor growth rate and change in immune status were observed.The subsets of the lymphocytes(CD4+T lymphocyte,CD8+ T lymphocyte,CD19+B lymphocyte)in peripheral blood and Treg lymphocyte in spleen of the three groups of mice were assayed with flowcytometry.Immune relate cytokines IL-2,IL-10,INF-? were measured with ELISA kit.At high tumor burden stage model,two groups of mice inoculated with hapa1-6 and when tumor formation were vaccine with embryonic stem cell or PBS.The tumor growth rate and change in immune status were observed.The subsets of the lymphocytes(CD4+T lymphocyte,CD8+ T lymphocyte,CD19+B lymphocyte)in peripheral blood and Treg lymphocyte in spleen and tumor tissues of the two groups of mice were assayed with flowcytometry.Immune relate cytokines IL-2,IL-10,INF-? were measured with ELISA kit.Results: At low tumor burden stage,tumor free rate of embryonic stem cell vaccine groups were significant difference with control group(p<0.001).Compared with control group,two vaccine groups showed significant difference in the subsets of the lymphocytes(CD4+T lymphocyte and CD8+T lymphocyte)in peripheral blood(P<0.0001)and significant difference in CD19+ B lymphocyte and Treg of spleen in vaccine intratumoral inject groups(p<0.0001).IL-2,INF-? in two vaccine groups showed significant difference with control group,and IL-10 only intratumoral inject groups showed significant difference with control groups(p<0.05).At high tumor burden stage,vaccine and control groups didn't show significant difference in tumor growth rate and tumor volumes at end of experiment.Two groups didn't show significant difference in the subsets of the lymphocytes and cytokines of peripheral blood.But at vaccine groups Treg increased in tumor tissue and showed significant difference with control group(p<0.05).Conclusion: Embryonic stem cell vaccine could inhibit liver cancer growth in mice at low tumor burden stage and vaccine intratumoral injection approach shows better immune regulated,but vaccine intratumoral injection approach can't boost immune to inhibit high tumor burden stage mice hepatoma.