Anti-tumor Effects Of HepG2 Tumor Vaccine Transfected HGM-CSF Gene Mediated By HA Nanoparticles

OBJECTIVE: Tumor recurrence and metastasis mainly due to the body's anti-tumor immune dysfunction resulting from, the performance of the immune escape and immune tolerance; tumor vaccine tumor antigen by enhancing immune function and reconstruction has become a cancer prevention A new therapy, hGM-CSF gene into the tumor cells can greatly increase the immunogenicity of cells. In this study, HA nanoparticles carried by hGM-CSF gene transfection, transfer prepared HepG2 hGM-CSF gene vaccine, and the transgenic HepG2 in vitro anti-tumor effect of the vaccine, and the gene-modified vaccine clinical application of liver cancer foundation.Method:1. Transgenic HepG2 cell vaccine preparation and identification of: HA nano-carriers carrying GM-CSF gene transfected HepG2 cells, RT-PCR method to detect GM-CSF gene mRNA, by ELISA assay hGM-CSF protein secretion, then the sub-lethal doses of radiation through after hGM-CSF prepared to carry genes HepG2 cell vaccine. Preparation of wild-type HepG2 same vaccine.2. Peripheral blood mononuclear cell separation and induced PBMC: peripheral blood from healthy people, isolated by density gradient centrifugation of peripheral blood mononuclear cells (PBMC), with different concentrations of transgenic and wild-type HepG2 HepG2 vaccine vaccine co-cultured Best selection induced concentration.3. PBMC group: According to the different cells will be induced PBMC divided into five groups of transgenic HepG2 vaccine, wild-type HepG2 vaccine group A, wild-type HepG2 vaccine group B, control group A, control group B, in which wild-type HepG2 vaccine A wild-type HepG2 as a simple vaccine induced, wild-type HepG2 wild-type vaccine group B vaccine plus hGMCSF factor-induced HepG2; blank control group A, group B add equal amount of control medium, IL-2 in the presence or absence of incubated under the conditions.4. PBMC of each group observed in vitro antitumor effect: Select the optimum induction concentration of PBMC in each group after the induction, flow cytometry analysis of CD4 +, CD8 + cell percentage changes, ELISA determination of INF-γsecretion, WST method in each group killing activity of PBMC.Results:1. Successfully prepared transgenic HepG2 cell vaccine and wild-type HepG2 vaccine. RT-PCR showed hGM-CSF gene was integrated in HepG2 cells and stable expression. ELISA detection of hGM-CSF gene into HepG2 cells secreting hGM-CSF, its secretion was 197.28±38.53ng/10E6cells every 24 h.2. Successfully separated PBMC, stained blue by Taiwan disc cell activity above 95%. In the I: E is 1:20, the transgenic and wild-type HepG2 HepG2 vaccine induced PBMC proliferation ability of the vaccine have the largest effect (P <0.05).3. I: E value is 1:20, flow cytometry showed that transgene PBMC in the vaccine group HepG2 percentage of CD4 + 57.49±0.98%, CD8 + cell percentage was 39.06±2.01; ELISA results showed that transgenic HepG2 in the vaccine group INF-γsecretion of PBMC amount of 1989.76±254.21pg/ml; WST showed PBMC transgenic HepG2 vaccine group than in the different conditions of effective target killing of HepG2 were 55.32±6.29,64.85±7.31 and 79.37±6.02, higher than the other group (wild-type HepG2 vaccine group A, wild-type HepG2 vaccine group B, control group A, control group B) PBMC (P <0.05)Conclusions:1. The use of HA nano-carriers to safely hGM-CSF gene into liver cells and stably expressed successfully prepared hGM-CSF gene transfected HepG2 cell vaccine.2. HepG2 cells transfected hGMCSF gene vaccine can effectively induce PBMC proliferation, differentiation and killing effects, treatment and prevention of HCC is expected to become new means of recurrence...