Određivanje upalnih citokina izlučenih iz THP-1 i NCI-H292 stanica nakon tretiranja izvanstaničnim Hsp70 proteinom

master's thesis

• Title (english): Measurement of pro-inflammatory cytokines secreted from THP-1 and NCI-H292 cells after treatment with extracellular Hsp70 protein
• Author: Ana Dojder
• Mentor: Lada Rumora (mentor)
• Committee member: Lada Rumora (predsjednik povjerenstva)
• Committee member: Marija Grdić Rajković (član povjerenstva)
• Committee member: Petra Turčić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2017-04-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Kronična opstrukcijska plućna bolest (KOPB) je bolest karakterizirana nedostatnim protokom
zraka kroz pluća pri čemu je više otežan izdisaj nego udisaj te je produljena ekspiracijska faza
respiracije. U patogenezi KOPB-a bitnu ulogu imaju oksidacijski stres i upalni procesi, jer do
opstrukcije dišnih putova dolazi zbog kronične upale dišnih putova i razaranja parenhima čime se
gubi alveolarna povezanost i elastičnost pluća. Glavni simptomi koji ukazuju da bolesnik boluje od
KOPB-a su: dispneja, kronični kašalj i kronično stvaranje sputuma, prisutnost KOPB-a kod drugih
članova obitelji te izlaganje rizičnim čimbenicima, odnosno štetnim česticama među kojima se
smatra da cigaretni dim ima najveću ulogu.
Cilj ovog istraživanja bio je ispitati ulogu izvanstaničnog rhHsp70 proteina te LPS-a i LTA u
imunosnom i upalnom odgovoru kod NCI-H292 i THP-1 staničnih linija te pri kojim
koncentracijama rhHsp70 protein uzrokuje statistički značajno povećano izlučivanje upalnih
citokina IL-1α, IL-1β, IL-6, IL-8 i TNF-α. Istraživanje je pokazalo da kod NCI-H292 stanica 0,3, 1
i 3 μg/mL rhHsp70 stimulira izlučivanje IL-1α i IL-8, dok 0,1, 0,3, 1, 3 i 30 μg/mL rhHsp70 potiče
otpuštanje IL-6. Osim toga, dokazano je kako tretiranje NCI-H292 stanica s LPS-om uzrokuje
povećano izlučivanje IL-6 i IL-8, te da LPS uzrokuje snažnije izlučivanje IL-6 i IL-8 nego LTA. S
druge strane, kod THP-1 stanica 10 i 30 μg/mL rhHsp70 uzrokuje povećanu koncentraciju IL-1β i
IL-8, ali ne i IL-1α, IL-6 niti TNF-α. Također, dobiveni rezultati pokazuju da THP-1 stanice
tretirane s LPS-om ili LTA izlučuju povećanu količinu svih ispitivanih citokina. Pri tome je
uočeno kako LPS ima izraženiji učinak od LTA i na THP-1 stanice.

Abstract (english)

Chronic obstructive pulmonary disease (COPD) is a disease characterized by insufficient flow of
air through the lungs wherein the exhalation is more difficult than the inhalation, and the expiratory
phase of respiration is prolonged. In the pathogenesis of COPD, oxidative stress and inflammatory
processes have an essential role due to chronic inflammation of the airways and destruction of
parenchyma which leads to lost of alveolar connectivity and lung elasticity. The main symptoms of
the patients suffering from COPD are dyspnea, chronic cough and chronic sputum production, the
presence of COPD at the other family members and exposure to risk factors or harmful particles
among which cigarette smoke plays a major role.
The aim of this study was to investigate the role of extracellular rhHsp70 protein, LPS and LTA in
the immune and inflammatory responses of NCI-H292 and THP-1 cell lines, and to find out which
concentrations of rhHsp70 protein cause statistically significant increased secretion of proinflammatory
cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α. Research showed that in NCI-H292
cells 0.3, 1 and 3 μg/mL rhHsp70 stimulates the secretion of IL-1α and IL-8, while 0.1, 0.3, 1, 3
and 30 μg/mL rhHsp70 provokes release of IL-6. Besides, our results have demonstrated increased
release of IL-6 and IL-8 from NCI-H292 cells treated with LPS, and this secretion was stronger if
compared with LTA treatment. On the other hand, in THP-1 cell line 10 and 30 μg/mL rhHsp70
caused increased concentrations of IL-1β and IL-8 in cell supernatants, but not of IL-1α, IL-6 or
TNF-α. Also, the results showed that THP-1 cells treated with LPS or LTA release increased levels
of all measured cytokines. Once again, LPS was stronger inducer of cytokines secretion than LTA.

• Keywords: KOPB; upalni citokini; Hsp70 protein; ELISA; LPS; LTA; TLR receptori; NCI-H292 stanice; THP-1 stanice
• Keywords (english): COPD; inflammatory cytokines; Hsp70 protein; ELISA; LPS; LTA; TLR receptors; NCI-H292 cells; THP-1 cells
• Language: croatian
• URN:NBN: urn:nbn:hr:163:535265
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-16 11:57:36