Agarose Encapsulation Of GM-CSF Genetically Modified Mouse Hepatoma Cells Restrict Growth Of Human Hepatoma Cells In Vitro

Hepatocellular carcinoma is one of the most important cancers that lead to humandeaths, with the increase in the incidence of factors, including viral infections,alcoholism, obesity, the incidence increased year by year. The malignancy ofhepatomacell is high, the progression is rapid, so a large number of the patients are diagnosedtoo late to get treatment.Conventional treatments including surgery, radiotherapy andchemotherapy, embolism have not obtainedcurative effect.Therefore people try toseek for a new therapy.In recent years, immunotherapy treatments of cancer areattaching people’s attention, the program includes a variety of liver cancerimmunotherapy: tumor cell vaccines, cytokine therapy, monoclonal antibody therapy,and adoptive immune cells treatment which includes:dendritic cells,lymphokine-induced tumor killer cells (CIK), cytokine activation of immune cells andtumor immunity gene therapy. These methods have made some progress inlaboratory and clinical applications.Studies suggest that inducting effective anti-tumor immunity needs two essentialelements in tumor tissue, first create a "danger signal" in tumor tissue, the death ofthe tumor cells not only provides the tumor antigens that the immune cells canrecognize but is also a danger signal while the immune response will beregulated byimmunomodulatory to produce anti-tumor immune suppressionwhich requires asecond factor at the tumor site with anti-tumor immune enhancement signal toreversethe antitumor immune that suppressed by the immunomodulatory. Currentimmunosuppressive therapy cannot satisfy both of the factors, this might be one of thefactors thatlead to poor anti-tumor effect of immunotherapy.To achieve the anti-liver cancer immunotherapy process while meeting the abovetwo factors, this study intends to conduct research through the following programs:(1) Application of hydrophilic agarose-embedded tumor cells for killing thetumor cells Research has proven hydrophilic agarose-embedded tumor cells can self-regulategrowth agarose beads embedded in giant form, secreting the substances that inducethe apoptosis of the cells. The secreting cells can also induce death of the co-culturedcells. By this way, agarose-embedded tumor cells constantly release tumor cell killingsubstances which lead to the death of the tumor cells and provide the danger signals.(2) Application of embeddedcells that secreting anti-tumor immune factors toprovide immune-enhancing signalIn this study, gene recombination and gene transfer technology will be applied totransfer the human granulocyte-macrophage colony-stimulating factor into theembedded tumor cells to express the factor stably.(3)Considering the growing tumor problem that the giant beads transplantingbrings, we use mouse cancer cells as seed cells that secret the tumor killing substances.At the same time, for regulating the side effect that the cytokine secretion brings,under the circumstances of being given pro drugs, the suicide gene is transferred intothe cell to kill the tumor cells in giant beads, for regulating the killing and immunereaction.Aiming at the above-mentioned research idea, the study use gene recombinationtechnologyand gene transfer techniques to form the mouse hepatoma cell that stablyexpress granulocyte macrophage colony-stimulating factor (GM-CSF) gene andherpes simplex virus (HSV-TK) and use the hydrophilic agarose to embed the genemodified mouse hepatoma cells to observe embedded cells to kill the hepatocellularcarcinoma of human and induce the anti-tumor immune.(1) Culture supernatant of hydrophilic agarose-embedded Hapa1-6cells inhibitthe growth of mouse Hapa1-6cells and HepG2human hepatoma cell lines.With the application of hydrophilic agarose Hapa1-6mouse hepatoma cellsentrapped, macrobeads formed. Collect the first3d,6d,12d,24d,35d culturalsupernatant, apply the cultural supernatant to the HepG2cells, test the activity byMTT on357d to estimate the inhibition of cell supernatants to the above-mentionedcells. The results show that the tumor cells are inhibited by supernatant of theembedded cells. This inhibition enhanced by time. Co-culture the giant beads embedded cells with HepG2cells, HepG2cells died after being co-cultured for5daysby microscope. This result further explained that the supernatant can inhibit the tumorcell growth.(2) Establish a mouse hepatoma cell lines stably expressing human GM-CSF andHSV-TK geneConstruct human GM-CSF eukaryotic expression vector HSV-TK gene by usingrecombinant technology. The two carriers have different selection marker. Take Hapa1-6as the research model, use the transfer technology mediated by liposome totransfer human GM-CSF into Hapa1-6cells. Screen the stably expressed cells bygiving puromycin. Test the level of gene transcription by RT-PCR technique and testthe GM-CSF in cell supernatants by ELISA. After establishing the GM-CSF genestably expressed cell lines, transfer the HSV-TK gene into the cells by liposome..Select the stably expressed cells by G418. Detect the HSV-TK gene expression levelof cell cloned by RT-PCR technique.Given prodrug GCV, cell death was observed.The result shows that GM-CSF and HSV-TK gene express stably in Hapa1-6cells,after giving GCV, cell death can be induced. Thus the mouse hepatoma cell lineswhich stably express GM-CSF and HSV-TK gene can be established.(3)A hydrophilic supernatant agarose coated Hapa1-6cells with anti-human liverbiology of tumorCulture the agarose-embedded normalhuman cells and mouse hepatoma cellswhich express GM-CSF and HSV-TK genes, collect the cell culturing supernatantsseparately. Co-culture the supernatants and human hepatoma cell lines. Estimate thegrowth of human hepatoma cell by MTT. The result shows that the supernatants caninhibit the growth of human hepatoma cell lines and the inhibition was enhanced bydays of embedding.(4) A hydrophilic agarose embedded GM-CSF gene-modified cells are capable oflong-term Hapa1-6GM-CSF protein secretionDetect the supertanant of GM-CSF-modified Hapa1-6cells cultured for differentembedded terms by ELISA. The result showed that the embedded cells decreased at3-12d and increased at12-35d, which showed that long-term secretion of cytokine and continuous immune stimulation were obtained by this means.(5) Pro-drugs of suicide gene can kill hydrophilic agarose-embedded cells.Culture the HSV-TK-modified Hapa1-6cells after being embedded byagarose,and give the pro-drugs of the suicide genes, detect the secretion of GM-CSF. Theresult showed that the secretion of GM-CSF decreased by time after given GCV(10ng/ml), but the secretion of GM-CSF without GCV didn’t change significantly.In conclusion,this study is based on the theory that effective tumor immunetreatment can be induced by the co-existence of death of tumor cells and immunologiccellular activity factor and the combination of genetic engineering techniques and cellembedding technology, and this study explored the solution that the death and thesecretion of immunologic cellular activity factor cannot co-exist in tumorimmunotherapy, which provides a new way for treatment of liver cancer.