Merjenje izražanja genov za imunosupresivne dejavnike mezenhimskih matičnih/ stromalnih celic kostnega mozga s potencialom za uporabo pri zdravljenju SARS-CoV2

Dolar Bratuša, Alisa (author), Zupan, Janja (mentor), Haring, Gregor (comentor)

Mezenhimske matične celice (MSC) so celice stromalnega izvora in so sposobne samoobnove in diferenciacije v celice mezodermalne plasti. Zelo pomembna lastnost MSC so njihove imunomodulatorne in protivnetne sposobnosti, ki pomagajo umiriti imunski odziv, s čimer prispevajo k popravilu tkiv in zmanjšanju nastajanja brazgotin. MSC so obetavne za zdravljenje različnih bolezni, vključno s COVID-19, zaradi svojih regenerativnih in imunomodulatornih lastnosti. Namen magistrske naloge je bil ugotoviti razlike imunosupresivnega potenciala treh vzorcev MSC po tretiranju z vnetnimi citokini. Testirane celice so bile pridobljene iz kostnega mozga mladega darovalca in predhodno gojene na dva načina, in sicer ročno ali v bioreaktorju Quantum. Kot kontrolne celice pa smo uporabili celice izolirane iz kosti stegnenice darovalca po smrti. Na podlagi zastavljenih ciljev magistrske naloge smo si postavili naslednje tri hipoteze. Prvič, da imajo testirane celice višje bazalno izražanje genov za vnetne dejavnike kot kontrolne celice. Drugič, da se po tretiranju z IL1β, IFN-γ in TNF-α poveča izražanja genov za vnetne dejavnike pri obeh vzorcih celic. Tretjič, da imajo testirane celice višje izražanje genov za vnetne dejavnike kot kontrolne celice po tretiranju z IL1β, IFN-γ in TNF-α. Primarne MSC smo namnožili v pogojih in vitro in jih nato tretirali s citokini IFN-γ, TNF-α in IL-1β. Po končanem tretiranju celic s citokini smo iz njih izolirali informacijsko RNA, ki smo jo nato prepisali v komplementarno DNA. Sledila je optimizacija metode kvantitativne verižne reakcije s polimerazo, ki smo jo uporabili za merjenje izražanja genov za vnetne dejavnike IDO1, TGFB1, CD274, TNFAIP6, IL1RN in HLA-G. Dobljene vrednosti smo normalizirali na referenčni gen GAPDH. Rezultati so pokazali, da se bazalno izražanje med testiranimi in kontrolnimi celicami razlikuje pri genih IL1RN, TNFAIP6 in HLA-G. Po tretiranju z IL1β, IFN-γ in TNF-α se poveča izražanje genov IDO1, TNFAIP6, IL1RN in HLA-G pri vseh treh vzorcih celic. Po tretiranju z IL1β, IFN-γ in TNF-α pa je izražanje genov TNFAIP6 in IL1RN višje pri testiranih celicah v primerjavi s kontrolnimi celicami. Tako smo vse tri hipoteze potrdili samo za gena IL1RN in TNFAIP6 ter s tem dokazali imunomodulatorni potencial testiranih celic, ki je osnova za njihovo nadaljnje testiranje pri boleznih kot je COVID-19.

• Language: Slovenian
• Keywords: mezenhimske matične celice, vnetni dejavniki, qPCR, izražanje genov, imunomodulatorne molekule
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-163031
• Publication date in RUL: 01.10.2024

Secondary language

• Language: English
• Title: Measurement of gene expression for immunosuppressive factors in bone-marrow-derived mesenchymal stem/ stromal cells with the potential for SARS-CoV2 treatment

Abstract

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.

• Keywords: mesenchymal stem cells, inflammatory mediators, qPCR, gene expression, immunomodulatory molecules