Povzetek
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.
Ključne besede
proteasom;imunoproteasom;izražanje genov;interferon-gama;imunski odziv;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2016 |
Tipologija: |
2.09 - Magistrsko delo |
Organizacija: |
UL FFA - Fakulteta za farmacijo |
Založnik: |
[K. Štrigl] |
UDK: |
577:616-097(043.3) |
COBISS: |
4042353
|
Št. ogledov: |
1487 |
Št. prenosov: |
344 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
ǂThe ǂanalysis of gene expression of the proteasome and immunoproteasome in different cell models |
Sekundarni povzetek: |
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. |
Sekundarne ključne besede: |
proteasome
immunoproteasome
gene expression
interferon-γ; |
Vrsta dela (COBISS): |
Magistrsko delo/naloga |
Komentar na gradivo: |
Univ. Ljubljana, Fak. za farmacijo |
Strani: |
VIII, 53 f. |
ID: |
12048483 |