Postavitev in optimizacija neradioaktivnega imunosupresivnega delovanja mezenhimskih matičnih celic in vitro : enoviti magistrski študijski program Farmacija

Grabnar, Bojana (author), Jeras, Matjaž (mentor)

Človeške mezenhimske matične celice (MMC) so sposobne diferenciacije v številne vrste somatskih celic, še zlasti tistih, ki so v vezivnem tkivu. V organizmu se nahajajo v različnih tkivih. Povečano zanimanje za njihovo klinično uporabo so spodbudila tudi spoznanja, da lahko vplivajo na imunske odzive v telesu, pri čemer je posebej pomembno njihovo zaviralno delovanje na aktivacijo limfocitov T. To pomeni, da bi z njihovo uporabo lahko preprečili nastanek akutne bolezni presadka zoper gostitelja (aGvHD) po presaditvi alogenskih KMC oz. jo na ta način zdravili. V okviru magistrske naloge smo si za cilj postavili vzpostavitev in optimizacijo neradioaktivnih proliferacijskih funkcijskih testov in vitro, ki bi nam omogočali zanesljivo določanje imunosupresivnega delovanja MMC. Najprej smo preskušali različne parametre gojenja in stimulacije mononuklearnih celic iz periferne krvi (MNC) in vitro, da bi ugotovili optimalen postopek neradioaktivnega določanja obsega T-celične proliferacije. Ugotovili smo, da je za izvedbo testa najprimernejša uporaba mikrotitrskih plošč s 96 vdolbinicami s polkrožnim dnom, brezserumskega medija BioTargetTM-1 ter 72-urna inkubacija MNC po njihovi aktivaciji z mitogenskim lektinom fitohemaglutininom (PHA) oziroma 6 dni po njihovi izpostavitvi aloantigenom v enosmernih in/ali dvosmernih mešanih limfocitnih kulturah (MLC). Obseg celične proliferacije smo vrednotili z merjenjem absorbanc po uporabi reagentov MTS (490 nm) ali CCK-8 (450 nm) in dodatni inkubaciji. Glede na rezultate smo kot najprimernejša testna modela izbrali mitogensko stimulacijo MNC s PHA in dvosmerno kulturo pomešanih MNC dveh nesorodnih oseb, ob uporabi reagenta MTS. V nadaljevanju smo ju nato uporabili za določitev obsega inhibicije T-celične proliferacije v prisotnosti različnih količin alogenskih MMC iz kostnega mozga. V primeru s PHA izzvane aktivacije, je bilo imunosupresivno delovanje MMC najmočnejše pri razmerju MNC : MMC = 8 : 1, v dvosmernih MLC pa pri razmerju MNC : MMC = 4 : 1. To razliko lahko pojasnimo z dejstvom, da alogenska stimulacija aktivira le omejeno število klonov limfocitov T, medtem ko mitogenska stimulacija s PHA izzove popoln proliferacijski odziv omenjenih celic.

• Language: Slovenian
• Keywords: mezenhimske matične celice akutna bolezen presadka mitogenska aktivacija limfocitov T alogenska aktivacija limfocitov T inhibicija T-celične proliferacije proliferacijski funkcijski testi
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [B. Grabnar]
• Year: 2018
• Number of pages: VII, 49 f.
• PID: 20.500.12556/RUL-120170
• UDC: 616-097+602.9(043.3)
• COBISS.SI-ID: 4459889
• Publication date in RUL: 16.09.2020

Secondary language

• Language: English
• Title: Estabishment and optimisation of an in vitro non-radioactive functional test for evaluation of immunosuppressive activity of mesenchymal stem cells : Uniform Master's Study Programme Pharmacy

Abstract

Human mesenchymal stem cells (MSCs) can differentiate into many types of somatic cells, especially those residing in connective tissues. These stem cells can be found in different parts of our bodies. Increased interest in their clinical use has been additionally promoted by the fact, that they can modulate human immune responses, particularly by inhibiting T cell activation. This means that by applying MSCs we could prevent or treat acute graft versus host disease (aGvHD) that can arise following allogeneic hematopoietic stem cells (HSCs) transplantation. In this study we set up a goal to establish and optimize a set of in vitro non-radioactive functional cell proliferation tests for evaluation of immunosuppressive activity of MSCs. First, we tested various parameters related to in vitro cultivation and stimulation of peripheral blood mononuclear cells (PBMCs) to determine optimal conditions for non-radioactive assessment of T-cell proliferation. We found that the use of 96 well (U-shaped) tissue culture plates, BioTargetTM-1 serum-free medium, as well as 72-hour incubation of PBMCs following their activation with phytohemagglutinin (PHA), and 6 days long incubation in case of their allogeneic stimulation induced in one- and two-way mixed lymphocyte cultures (MLCs), are optimal for performing our tests. The extents of T-cell proliferation were assessed by measuring absorbances after the addition of MTS or CCK-8 reagents to cell cultures, followed by additional incubation. According to results we decided to use the MTS reagent and to select mitogenic (PHA) stimulation of PBMC, as well as the two-way MLC, set-up by mixing PBMCs of two unrelated donors, as the two most appropriate test models. Further on we applied both of them to evaluate the extent of T-cell proliferation inhibition caused by the presence of different amounts of allogeneic bone-marrow-derived MSCs. In case of PHA-induced proliferation of T lymphocytes the immunosuppressive action of MMCs was the greatest at the PBMC : MMC ratio of 8 : 1, while in the two-way MLCs such effect was observed at the PBMC : MMC ratio of 4 : 1. This difference could be a consequence of the fact that allogeneic stimulation only activates a limited number of T cell clones, while the mitogenic lectin (PHA) T-cell activation is complete.

• Keywords: mesenchymal stem cells (MSCs) acute graft versus host disease (aGvHD) mitogenic and alloantigenic activation of T cells inhibition of T-cell proliferation non-radioactive proliferation functional tests