| dc.description.abstract | Immune checkpoints are one of the methods of modulating the immune system. Some cancer cells, by increasing the production of these immune checkpoints, reduce the power of immune cells and T lymphocytes in destroying tumor cells. Tomozolomide (TMZ) is considered first-line therapy for the treatment of newly diagnosed glioblastoma. The purpose of this study is to investigate the effect and role of temozolomide drug in activating or inhibiting dendritic cells as well as the level of expression of immune checkpoints, so that if possible, this drug can be used in immunotherapy methods and it can be used as a new treatment. , considered less complicated and effective.
Methods: First, peripheral blood mononuclear cells (PBMCs) were isolated using Faycol solution. Then, monocytes were separated from PBMC through their adhesion to polystyrene surfaces, and using IL-4 and GM-CSF cytokines, monocyte-derived DCs were produced. Differentiation, maturation and activation of dendritic cells were measured by flow cytometry. To examine DCs, in addition to the morphological examination of these cells during differentiation during the cell culture process by means of images taken with an Optika light microscope, the expression of various surface markers expressed on these cells was also used. Expression of markers such as CD11c, HLA-DR, CD86 and CD40, using anti-CD11c-FITC, anti-HLA-DR-APC, anti-CD86-PerCP-cy5.5 and anti-CD40-CF-blue antibodies by flow cytometry Done. After phenotyping, DCs were incubated with LPS to mature and activate. Different doses (50, 500 and 1000 μM) were investigated to obtain the best dose for temozolomide. Then, to check the amount of apoptosis and necrosis induced by different doses, annexin and PI were used respectively and read by flow cytometry. Finally, to measure the effect of mDC on the performance of temozolomide drug, mDCs were treated with temozolomide drug and the relative expression of PD-L1, CTLA-4, LAG3, TIM3, BTLA and VISTA genes was obtained by Real Time PCR method.
Results: The results obtained from flow cytometry expression of surface markers CD11c, HLA-DR, CD86 and CD40 on the cells showed that DC cells were well derived from monocytes. Also, according to the results of apoptosis and liver necrosis flow cytometry, the best effective dose of temozolomide on mDC was determined to be about 500 μM. The expression level of PD-L1, CTLA-4, LAG3, TIM3, and VISTA genes in the TMZ-mDC group has decreased significantly compared to the mDC group. Meanwhile, BTLA gene expression in TMZ-mDC group did not change significantly compared to mDC group. | en_US |
