Mesenchymal stem cells (MSCs) are stromal cells capable of self-renewal and differentiation into cells of the mesodermal lineage. A significant characteristic of MSCs is their immunomodulatory and anti-inflammatory abilities, which help to calm the immune response, thereby contributing to tissue repair and reducing scar formation. Due to their regenerative and immunomodulatory properties, these cells hold promise for the treatment of various diseases, including COVID-19. The aim of the master's thesis was to determine the differences in the immunosuppressive potential of three MSC samples after treatment with inflammatory cytokines. The tested cells were derived from the bone marrow of a young donor and previously cultured in two ways: manually or in a Quantum bioreactor. As control cells, we used cells isolated from the femur bone of a deceased donor. Based on the set objectives of the master's thesis, we established the following three hypotheses. First, that the tested cells have a higher basal expression of inflammatory factor genes compared to the control cells. Second, that the expression of inflammatory factor genes increases in both cell samples after treatment with IL1β, IFN-γ, and TNF-α. Third, that the tested cells have higher expression of inflammatory factor genes compared to the control cells after treatment with IL1β, IFN-γ, and TNF-α. Primary MSCs were expanded under in vitro conditions and then treated with the cytokines IFN-γ, TNF-α, and IL-1β. After the cytokine treatment, we isolated messenger RNA from the cells, which was then transcribed into complementary DNA. This was followed by the optimization of the quantitative polymerase chain reaction method, which we used to measure the expression of the inflammatory factor genes IDO1, TGFB1, CD274, TNFAIP6, IL1RN, and HLA-G. The obtained values were normalized to the reference gene GAPDH. The results showed that basal expression differed between the tested and control cells in the genes IL1RN, TNFAIP6, and HLA-G. After treatment with IL1β, IFN-γ, and TNF-α, the expression of the genes IDO1, TNFAIP6, IL1RN, and HLA-G increased in all three cell samples. After treatment with IL1β, IFN-γ, and TNF-α, the expression of the genes TNFAIP6 and IL1RN was higher in the tested cells compared to the control cells. Thus, we confirmed all three hypotheses only for the genes IL1RN and TNFAIP6, thereby demonstrating the immunomodulatory potential of the tested cells, which forms the basis for their further testing in diseases such as COVID-19.
|
