The Anti-tumor Effects Of DC Vaccine Loaded With HBx Antigen In Vivo Of Human Immune Reconstituted Nude Model With Human Hepatoma

Objective:To investigate the anti-tumor effect of DC vaccine loaded with HBx antigen onhuman immune reconstitution in nude mice bearing human hepatoma model invivo,which providing new and improved ways and scientific basis for immune therapyof hepatoma.Methods:1.Identification and culture of HBx protein stably transfected HepG2humanhepatoma cell line: the use of RT-PCR and Western blot assay to detect the expressionof HBx protein in stably transfected HepG2human hepatoma cell lines HBx protein;determine the G418selection concentrations of stably transfected cell lines.2.Establishment and Identification of immune reconstitution Nude Model:12noimmune leakage mice were randomly divided into2groups:Intraperitoneal injection ofhuman peripheral blood mononuclear cells in6mice as a human immune reconstitutiongroup, Intraperitoneal injection of sterile PBS only in6mice as a the control group.Observed the biological characteristics of mice; ELISA detection of human IgG levelsin peripheral blood of mice; HE staining was used to observe whether mice liver andspleen tissue have infiltration of lymphocytes.3. The anti-tumor effects of DC vaccine loaded with HBx antigen in vivo ofhuman immune reconstituted nude model with human hepatoma: DC cells were inducedby cell factors (GM-CSF, IL-4, and TNF-α)from human peripheral blood mononuclearcells in vitro.Prepare DC vaccine loaded with HBx antigen.After24hours of reconstituted human immune，15nude were seeded with stably transfected HBx antigenHepG2cells,and were devided radomly into3groups in7days: HBx-DC group,5mice,each intraperitoneal injection HBx antigen pulsed DC vaccine2x105/0.2ml; DC group,5mice, per only intraperitoneal injection DC2x105/0.2ml; the control group,5mice,each intraperitoneal injection of saline0.2ml. Observe the growth of mice and tumortissue; ELISA assay was used to detect the TH1, TH2type cytokines expression inmice serum; PI staining were uesd to observe the apoptosis of hepatoma cell lines,andcalculated tumor cell proliferation index; Observed the parhologic changes of the tumorrissues;Immunohistochemistry analysis were used to detect the expression of apoptosisrelated proteins(P53、Bax、Casepas-3、Bcl-2、VEGF、CD34、MMP-2、CDK2、MMP-9、P21)in tumor tissues.Results:1.RT-PCR detection of cell lines shown that the expression of HBx mRNA instably transfected with HBx group,was significantly higher than the control group andthe empty vector group, and the difference was statistically significant (P <0.05).Western blot detection of cell lines shown that HBx protein expression of the stablytransfected with HBx group were higher than the others and the difference wasstatistically significant (P <0.05). After two weeks observation,concentration of600μg/ml can cause cells floating death, that is, the cell line screening concentration.2.None of the immune reconstitution nude was died in9weeks, and nograft-versus-host reactions was detected. Respectively in2,4,6,8weeks after injection,ELISA assay shown that the human IgG levels in nudes’ peripheral blood of mice inimmune reconstitution group was graduately advaced and was significantly higer thanothers at the same point(P <0.05). HE staining of mice liver and spleen tissues foundthat their have infiltration of lymphocytes in spleen tissues of human immunereconstitution nude.3. The mice of HBx-DC group had a good mental state while the control groupacted listlessness, local tumor tissue occured ulcerations. Subcutaneous tumor growth inthe HBx-DC group was significantly slowed down, and the difference was statistically significant (P <0.05) after one week. The mice were sacrificed six weeks, weighed,thetumor tissue,and found that the tumor weight in the HBx-DC group was significantlylower than the other two groups, the difference was statistically significant (P <0.05),the tumor inhibition rate was0.6. In the5th week of treatment, ELISA assay was found:In the HBx-DC group,Th1type cytokines(IL-2, IFN-γ, TNF-α) increased significantly,Th2cytokines(IL-4, IL-6, IL-10)significantly decreased, the differences werestatistically significant (P <0.05). The tumor cell proliferation index in the HBx-DCgroup was significantly lower than the other two groups (P <0.05). Tumor pathologyconfirmed for hepatoma with HBx expressed in mice, and the tumor tissues in HBx-DCgroup occured hemorrhage, necrosis,and had a large number of lymphocytes infiltration.Immunohistochemistry found in HBx-DC group, expression of P53, Bax, Casepas-3,P21was significantly upregulated (P <0.05), expression of Bcl-2、VEGF、CD34、MMP-2、MMP-9expression was significantly lower (P <0.05).Conclusion:1.HepG2human hepatoma cell lines stably expressing HBx protein2.Immune reconstitution nude model successfully establisherd by intraperitonealinjection of human peripheral blood lymphocytes,which can better simulate the humanimmune environment, providing an animal model for the further study ofimmunotherapy.3. The DC vaccine loaded with HBx antigen can significantly slow down tumorgrowth, regulate immune balance, inhibite tumor cell proliferation and promotedtumorcell necrosis, enhance anti-tumor immunity in immune reconstitutied bearing humanhepatoma nude model in vivo.