The Study On The Anti-Gastric Cancer Effects And Mechanism Of B7-H1 Antisense RNA Modified Dendirtic Cells Vaccine

Background: Gastric cancer is one of the most common malignant gastrointestinal diseases in many countries .Treated with curative operation, chemotherapy and radiotherapy, most patients had to face up to a poor prognosis. Immunotherapy is one of the new methods for anti-tumor therapy. Moreover, how to activate enough T cells is one of the most important elements. Dendritic cells(DCs), could activate naive T cell and induce antigen specific CTL, play a major role in the generation of immunity against tumor cells. Which had been demonstrated in many animal and clinical experiments. Those researches, however, mainly focused on how to enhance the ability of DCs received and processed information that could activated T cells. Researchers noticed some molecules expressed on the surface of DCs in recent years. Because those molecules could regulate the function of DCs. B7-H1 is a recently identified member of the B7 family molecules. Which is abundant in various human cancers. Current data suggest that B7-H1 could inhibit the function of T cells. The more recently described that B7-H1 also expressed on the surface of DCs, especially on the mature DCs. Whether B7-H1 expression could affect the biological function of DCs and inhibit to transfer the information of activating T cell. In this experiment, we construct the adenoviral vector containing B7-H1 antisense gene and transfect the vector into DCs in vitro. To investigate the anti-gastric cancer effects and mechanism of B7-H1 antisense RNA modified DCs vaccine. We hope to provide a theoretical basis and a new method for clinical application of DCs vaccine .Method: Part Ⅰ.Recombinant Adnovirus were generated by liposome cotransfected of pDC315.ASB7-H1 with adenovirus genomic plasmid pBHGloxΔE1,3cre in 293 cell. The viral stocks were generated in 293 cells and concentrated by cesium chloride ultracentrifugation. An Ad vector expressing the bacterial β -galactosidase gene (Ad.LacZ) was amplified and purified and used as controls. The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. Part Ⅱ.Peripheral blood mononuclear cells were cultured with GM-CSF ,IL-4 and TNF- α to induced DCs,After the recombinant adenovirus infected DCs, Antisense RNA and protein expression of B7-H1 were evaluated by RT-PCR and Western blot analysis. The altered phenotypes of dendritic cells and the Ad.ASB7-Hl transfected DCs were detected by flow cytometry in order to evaluated the effects of B7-H1 antisense RNA on phenotype of DCs. Part HI. 3H-TDR was used to detect the capacity of the Ad.ASB7-Hl transfected DCs to activate T lymphocyte proliferation. Enzyme-linked immunosorbent assay (ELISA) method were used to detect IL-12 level of supernatant, 51Cr release test was performed to evaluate the gastric cancer cell antigen specific CTL response induced with Ad.ASB7-Hl transfected DCs. We also analyzed the cycle of gastric cancer cells after treated with Ad.ASB7-Hl transfected DCs vaccine with the flow cytometry. Part IV .ELISA detected the IL-10, interferon-y (IFN-y) which was secreted by activated T lymphocytes. Finally, the flow cytometry was used to detected the rate of T cell activation after stimulated by Ad.ASB7-Hl transfected DCs, The rate of apoptosis of activated T cell and alteration of its expression of FasL, too. Results: ?A replication-deficient adenovirus type 5 carrying B7-H1 antisense gene was constructed via site special recombination in 293 cells. The recombinant adenovirus proved correct by PCR. Plaque titration on 293 cells showed titers of 5X 1010pfu/ml. (2) DCs had been induced from healthy doners peripheral blood monocyte cells with IL-4, GM-CSF,TNF- a .Those DCs had typical morphological characteristics and phenotype. The expression of B7-H1 gradually increased in the course of maturation and could reach above 90% at last. ?The expression of B7-H1 antisense RNA was very obvious in DCs after transfected by recombinant adenovirus Ad.ASB7-Hl. Ad.ASBB7-Hl transfection significantly inhibited B7-H1 mRNA and protein expression induced by cytokines. @ Ad.ASB7-Hl transfection significantly increased the potency of secretion of IL-12 by transfected DCs and enhanced the ability of DCs to stimulate proliferation of T cell. ?The effectors T cell induced by the Ad.ASB7-Hl transfected DCs loaded with gastric antigen had more obviously cytotoxic effects to tumor cells than control groups. Moreover, the Ad.ASB7-Hl transfected DCs vaccine could inhibited the progress of gastric cancer cell cycle and induced it apoptosis.?The Ad.ASB7-Hl transfected DCs could stimulate more T cell express CD25 and CD69 than the Ad.ASLacZ transfected DCs and normal control DCs(87.61±6.46%, 89.65 + 6.92% vs. 72.43 + 3.86%, 72.43±3.86% and 74.18 + 4.30% , 70.27+4.89%respectively, P<0.05). The Ad.ASB7-Hl transfected DCs also couldinhibited FasL expression on activated T cell and reduced apoptosis of activated T cell(74.73 + 7.96% vs. 89.89 + 8.14%, 48.45 + 3.78% vs. 66.48 + 6.15 % respectively, p< 0.05) .?The T cell producted more IFN- Y and less IL-10 after stimulated by Ad.ASB7-Hl transfected DCs(2764 + 212.43pg/ml vs. 1736 + 182.31pg/ml , 168±19.37pg/ml vs. 478±62.79pg/ml respectively, p<0.01 )?Conclusion: (￡>A recombinant replication adenovirus Ad.ASB7-Hl was constructed in this experiment. The adnovirus vector could amplify in 293 cells, it tillers could reach 5 X 1010/pfu/ml. ?The expression of B7-H1 antisense RAN was steady in DCs after transfected by recombinant adenovirus vector, which could significantly inhibit the expression of B7-H1 mRNA and protein. Moreover, it also inhibited the expression of B7-H1 on the surface of DCs. (3)DCs could secrete more IL-12 and enhance the activity of immunostimulation after modified with B7-H1 antisense RNA. Ad.ASB7-Hl transfected DCs could inhibit the proliferation of gastric cancer, induce apoptosis of gastric cancer and enhance the effect of anti-gastric cancer. ?The main mechanism of antigastric cancer induced by Ad.ASB7-Hl transfected DCs as follows. Induced activation of T cell efficiently, inhibited apoptosis of activated T cell ,downregulated the expression of FasL on the activated T cells and derived profile of cytokine secretion to Thl mode. (5) Ad.ASB7-Hl transfected DCs vaccine might provide a new kind of biotherapy means for gastric cancer in clinical application.