| Backgrounds: In recent years, there is a ascendent tendency with NHL in incidence rate and mortality rate . Due to the application of chemotherapy and radiotherapy ,the 5 years survival rate of NHL has rised from 30% in 1970's to 52% nowadays. Nevertheless,the traditional treatment modes are harmed to normal tissue.Furthermore, it is difficult to annihilate tumour cells thoroughly ,MRD is usually the major cause for NHL resistant to drugs and relapse. Therefore, new therapeutic techniques are needed for combating this disease.As a new kind of treatment mode ,the Management of biology, which has some advantages such as target treatment, trace residual of tumour cells in vivo and little side effect. Recent studies have focused on the immunotherapy for malignancies mediated by DCs (dendritic cells) since DCs can be heavy amplificated in vitro.NHL is suitable for immunotherapy because of the expressing of tumor specific antigen(TSA) and tumor associated antigen(TAA). As an important cytokine for antigen presenting cell(APC), Granulocyte-macrophage colony stimulating factor(GM-CSF) has the capacity to promote and to keep maturation of DCs ,up-regulate the surface expression of costimulatory molecules.Therefore , we plan to induce NHL CA46 specific CTLs induced by DCs in vitro, which pulsed with CA46 cell line antigens and transfected with recombinant adenovirus vector containing human GM-CSF gene,and assess the lethal effect of cytotoxic lymphocyte against CA46 cells.Objective: The goal of the research was to assess the lethal effect of cytotoxic lymphocyte against CA46 cells induced by DCs ,which is transfected with GM-CSF recombinant adenovirus and pulsed with CA46 lysate.Methods:The PBMCs were isolated by density gradient centrifugation and monocytes were received by adhesion to plastics.The monocytes were cultured at 37℃in a CO2 incubator. GM-CSF and IL-4 were added per 3 days, while TNF-αwas added on 7th day.Then,these cells were harvested identified for morphology characteristics by using phase-contrast microscopy and electronmicroscope. The immunophenotypy of these cells were analysed by flow cytometry and T cell stimulatory ability by mixed lymphocyte reaction (MLR) test. The amplification of a virus stock is achieved by infection of Ad-293 cultures with the primary viral stocks.The cytotoxic lymphocyte against CA46 cells were induced by cultureing with DCs for 5 days, which pulsed with CA46 antigens and transfected with GM-CSF recombinant adenovirus . The cytotoxic lymphocyte against CA46 cells response was measured by LDH release detection and interferon-γproduction was also tested by ELISA ( enzyme-linked immunosorbent assay).There are three groups in the study : antigenic stimulation and gene transfection group(C-G-DC group), antigenic stimulation group (C-DC group),pure DC group (N-DC group).Result: Typical DCs were generated from peripheral blood monouclear cells (PBMC) by cultured with GM-CSF,IL-4 and TNF-α.Except for CD80, they expressed CD83,CD86,HLA-DR highly on their surface ,and seldom expressed CD14. The expression of GM-CSF was confirmed by RT-PCR .Specific killing of CA46 cells mediated by CTLs were tested with LDH assay. At 5:1 of effector/target ratio, the killing rate of C-G-DC group,C-DC group and N-DC group was 21.5%±1.9%,,8.1%±1.1% and 1.8%±0.3%, respectively. At 10:1 of effector/target ratio, the killing rate of the three groups was 49.1%±4.2%,17.9%±1.3% and 5.4%±0.5%,respectively.At 20:1 of effector/target ratio, the killing rate was 63.6%±6.3%,32.4%±5.7%and 6.7%±0.6%,respectively.We found the killing rate of C-G-DC group analyzed by the statistics software SPSS13.0 was the highest,and the level of IFN-γwas also the highest among the three groups.Conclusion:PBMC can be cultivated to mDCs with the cytokine of GM-CSF,IL-4 and TNF-α;The cytotoxic lymphocyte against CA46 cells can be induced by DCs pulsed with CA46 lysate ,and the lethal effect by CTLs can be enhanced when DCs were modified by GM-CSF gene.
|
PDF Full Text Request