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Diferenciacija limfocitov T s stresnimi zunajceličnimi vezikli
ID Erjavec, Klavdija (Author), ID Benčina, Mojca (Mentor) More about this mentor... This link opens in a new window, ID Mancek Keber, Mateja (Co-mentor)

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Abstract
Človeški organizem se na oksidativni stres, ki poganja sterilno vnetje, odzove z mehanizmi prirojene imunosti, ki jih sproži prepoznavanje molekul oziroma strukturnih motivov, značilnih za poškodovano tkivo (DAMP). Posledica tega odziva je tudi sproščanje stresnih zunajceličnih veziklov (sEV), glavnih akterjev medceličnega komuniciranja, preko katerih celice odstranjujejo oksidirane molekule. Na ta način lahko usmerjajo tudi pridobljeni imunski odziv v smeri različnih efektorskih povezav limfocitov T. Slednje je odvisno predvsem od ustrezne aktivacije in zorenja dendritičnih celic. V obsegu magistrske naloge smo zato želeli ovrednotiti vpliv sEV na diferenciacijo limfocitov T, predvsem preko IL-23/Th17 poti. Čeprav endogeni ligandi delujejo preko istih receptorjev kot ligandi pri mikrobih, smo ugotovili, da sEV sprožijo drugačen imunski odziv kot LPS, kar potrjujejo tudi razlike v njunem signaliziranju. S pomočjo kvantitativnega PCR smo določili izražanje mRNA provnetnih citokinov, predvsem I l23, kot tudi drugih citokinov, po stimulaciji dendritičnih celic s sEV. Naivne limfocite T CD4+, ki smo jih izolirali iz vranice, smo stimulirali s kokulturo dendritičnih celic, ter spremljali koncentracijo citokinov IL-17 in IL-2 s testom ELISA. Rezultati raziskave so pokazali, da količina citokinov, ki se je izločila iz dendritičnih celic, ni zadostovala za uspešno diferenciacijo limfocitov T v tip Th17 in ne v tip Th1. Dodatna stimulacija s sEV in daljši čas gojenja v kokulturi sicer privedeta do izločanja IL-17, vendar premalo, da bi lahko govorili o uspešni diferenciaciji tipa Th17. Natančen molekularni mehanizem delovanja sEV na diferenciacijo limfocitov T tako ostaja deloma neraziskan. V prihodnje bodo za njegovo razjasnitev potrebne dodatne raziskave, predvsem v smislu aktivacije samega mehanizma, posledic aktivacije v odnosu do kroničnih bolezni, ter načinov njegove inhibicije.

Language:Slovenian
Keywords:imunologija, oksidativni stres, zunajcelični vezikli, BMDC, IL-23, diferenciacija, limfociti Th17, IL-17
Work type:Master's thesis/paper
Organization:BF - Biotechnical Faculty
Year:2020
PID:20.500.12556/RUL-117783 This link opens in a new window
Publication date in RUL:26.07.2020
Views:1072
Downloads:195
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Secondary language

Language:English
Title:DIFFERENTIATION OF LYMPHOCYTES T WITH STRESS EXTRACELLULAR VESICLES
Abstract:
The human body responds to oxidative stress, a driver of sterile inflammation, by using the mechanisms of innate immunity, through the recognition of damage-associated molecular patterns (DAMP). During oxidative stress cells remove oxidized molecules by releasing stress derived extracellular vesicles (sEV). sEVs mediate intercellular communication. In this way they can also drive adaptive immune response in the direction of various T lymphocytes effector cells. The latter depends mainly on the account of consistent activation and maturation of dendritic cells. In the scope of the master's thesis, we therefore wanted to evaluate the influence of sEV on the differentiation of T lymphocytes, especially via the IL-23/Th17 pathway. Although endogenous ligands act through the same receptors as ligands in microbes, they elicited a different immune response, as shown by differences in LPS signaling versus sEV. After stimulation of dendritic cells with sEV by using qPCR we determined mRNA expression of proinflammatory cytokines, primarily Il23, as well as other cytokines. Naive T CD4 + lymphocytes isolated from the spleen were stimulated by dendritic cell culture, and the concentration of cytokines IL-17 in IL-2 was monitored by ELISA. The results of the study showed that the amount of cytokines secreted from dendritic cells was not sufficient for successful differentiation of T lymphocytes into Th17 type or Th1 type. Repeated stimulation with sEV and longer cultivation time in co-culture slightly increased the secretion of IL-17, but was not enough to show a successful differentiation of Th17 cells. The exact molecular mechanism of action of sEV on T lymphocyte differentiation still remains partially unknown. In the future, additional research will be needed to clarify this mechanism, especially in terms of its activation, the consequences of activation in relation to chronic diseases, and ways to inhibit it.

Keywords:imunology, oxidative stress, extracellular vesicles, BMDC, IL-23, differentiation, lymphocytes Th17, IL-17

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