Analiza izražanja genov proteasoma in imunoproteasoma v različnih celičnih modelih

Štrigl, Kaja (avtor), Mlinarič-Raščan, Irena (mentor), Šmid, Alenka (komentor)

Patologija kroničnih, nevrodegenerativnih in malignih obolenj je povezana z molekulami proteasoma in imunoproteasoma, ki so predmet raziskav kot tarče za nove terapevtske pristope. Proteasomi imajo velik pomen za uravnavanje znotrajceličnih funkcij in funkcij imunskega sistema, saj razgrajujejo regulatorne proteine in proizvajajo antigenske peptide za predstavitev imunskim celicam. Namen našega dela je bil ovrednotiti izražanje genov, ki kodirajo podenote proteasoma in imunoproteasoma. Želeli smo doprinesti k razumevanju funkcij proteasoma in imunoproteasoma ter tudi na izbrani celični liniji Thp1 analizirati izražanje genov po tretmaju z interferonom-γ, saj predhodne študije dokazujejo, da inducira izražanje imunoproteasomskih podenot. Eksperimentalno delo smo opravili na 16 vzorcih, ki so zajemali imunske celice in celice drugih tkiv. Za proučevanje izražanja genov smo uporabili metodo qPCR, ki smo jo morali najprej optimizirati, nato pa smo z njo izmerili izražanje preiskovanih genov ter s pomočjo standardne krivulje izračunali relativno koncentracijo mRNA posameznega gena v vzorcih. Vsak gen za posamezni vzorec smo normalizirali na dva referenčna gena (GUSB, RPLP0), ki smo jih predhodno izbrali s pomočjo algoritma NormFinder®, ki izračuna koeficient stabilnosti kandidatnih genov. Analizirali smo nivo izražanja genov, ki kodirajo podenote proteasoma β1 (PSMB6), β2 (PSMB7) in β5 (PSMB5) ter genov, ki kodirajo podenote imunoproteasoma β1i (PSMB9), β2i (PSMB10) in β5i (PSMB8). Ugotovili smo statistično značilno večje izražanje genov, ki kodirajo podenote imunoproteasoma pri imunskih celicah, medtem ko je bilo izražanje genov, ki kodirajo podenote proteasoma pri imunskih celicah nižje. Največje statistično značilno izražanje genov, ki kodirajo podenoti proteasoma β1 in β2 je bilo pri celicah Jurkat (akutna T celična levkemija). Pri celicah Thp1 je prišlo do najmanjšega izražanja genov, ki kodirajo podenoto proteasoma β1. Pri imunoproteasomu je prišlo do najvišjega izražanja genov, ki kodirajo podenoto β1i v limfoblastoidni celični liniji LCL5. Pri podenoti β5 je prišlo v primerjavi s podenoto β5i do statistično značilnega večjega izražanja genov v imunskih celicah in tudi pri celicah drugih tkiv (HepG2). Izražanje genov, ki kodirajo β5i podenoto je bilo največje pri celični liniji NCTC2544, najmanjše pa pri celični liniji MCF7. Zanimalo nas je tudi, kako vnetni faktor interferon-γ za katerega je dokazano, da poveča izražanje genov, ki kodirajo podenote imunoproteasoma, vpliva na izražanje genov pri izbrani imunski celični liniji Thp1. Zato smo nazadnje celice tretirali v različnih časovnih okvirjih (24, 48 in 72 ur) ter nato primerjali tretirane in netretirane celice. Naš rezultat je pokazal, da se po tretmaju (72 ur) statistično značilno poveča izražanje genov, ki kodirajo podenoto β1. Spremembe v izražanju za podenoti β2 in β5 pa statistično niso bile značilne v primerjavi s kontrolno skupino. Ugotovili smo, da je imel tretma celic z interferonom-γ večji učinek na izražanje genov, ki kodirajo vse podenote imunoproteasoma (β1i, β2i in β5i). Dokazali smo, da obstajajo statistično značilne razlike med tretiranimi in netretiranimi celicami.

• Jezik: Slovenski jezik
• Ključne besede: proteasom imunoproteasom izražanje genov interferon-gama imunski odziv
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [K. Štrigl]
• Leto izida: 2016
• Št. strani: VIII, 53 f.
• PID: 20.500.12556/RUL-81484
• UDK: 577:616-097(043.3)
• COBISS.SI-ID: 4042353
• Datum objave v RUL: 11.04.2016

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The analysis of gene expression of the proteasome and immunoproteasome in different cell models

Izvleček

Pathology of chronic, neurodegenerative and malignant diseases is associated with molecules of proteasome and immunoproteasome, which are the subject of researches as targets for new therapeutic approaches. Proteasomes are of great importance due to their regulation of intracellular functions and functions of the immune system; in other words, regulatory proteins are degraded and antigenic peptides for presentation of immune cells are produced. The purpose of our work was to evaluate the expression of genes which encode the subunits of proteasome and immunoproteasome. We wanted to contribute to the understanding of functions of proteasome and immunoproteasome and also to analyse gene expression after the treatment with interferon-γ on the selected cell line Thp1, as the previous studies have shown that interferon-γ induces the expression of subunits of immunoproteasome. Experimental work was implemented on 16 samples, which included immune cells and cells from other tissues. The qPCR method was used in order to study the gene expression and was optimized prior to conducting the experimental work. By using this method the expression of investigated genes was measured and the relative concentration of mRNA of each gene in the samples was calculated with the help of the standard curve. Each gene for each sample was normalised onto two reference genes (GUSB, RPLP0) which were previously selected with the help of NormFinder® algorithm that calculates the coefficient of stability of candidate genes. The level of expression of genes which encode the subunits of proteasome β1 (PSMB6), β2 (PSMB7) and β5 (PSMB5) and the subunits of immunoproteasome β1i (PSMB9), β2i (PSMB10) and β5i (PSMB8) was analysed. A significantly higher expression was found in genes which encode the subunits of immunoproteasome in immune cells, whereas the expression of genes which encode the subunits of proteasome in immune cells was reduced. The significantly highest expression of genes which encode the subunits of proteasome β1 and β2 was found in Jurkat cells (acute T cell leukemia). The lowest expression of genes which encode the subunit β1 of the proteasome was found in Thp1 cells. The highest expression of genes which encode the subunit β1i of the immunoproteasome was discovered in LCL5 lymphoblastoid cell line. The gene expression in immune cells and also in cells from other tissues (HepG2) was significantly higher in the subunit β5 in comparison to the subunit β5i. The highest expression of genes which encode the subunit β5i was found in NCTC2544 cell line and the lowest in the MCF7 cell line. The question was also arisen on how proinflammatory factor interferon-γ, which has been shown to increase the expression of genes which encode the subunits of immunoproteasome, affects the expression of genes in the selected Thp1 immune cell line. Finally, the cells were being treated in various time frames (24, 48 and 72 hours) and the comparison of treated and untreated cells was conducted. The result has shown that the expression of genes which encode the subunit β1 was significantly higher after the treatment (72 hours). The changes in the expression for subunits β2 and β5 were not statistically significant in comparison to the control group. It has been established that the treatment with interferon-γ had a greater effect on the expression of genes which encode all subunits of the immunoproteasome (β1i, β2i and β5i). It has been proven that there are statistically significant differences between treated and untreated cells.

• Ključne besede: proteasome immunoproteasome gene expression interferon-γ