Utjecaj agonista TLR receptora na vijabilnost NCI-H292 epitalnih stanica

master's thesis

• Title (english): Effect of TLR receptor agonists on NCI-H292 cell viability
• Author: Lucija Sladoljev
• Mentor: Lada Rumora (mentor)
• Committee member: Lada Rumora
• Committee member: Ana-Marija Domijan
• Committee member: Anita Somborac Bačura
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2015-07-09, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Kronična opstrukcijska plućna bolest karakterizirana je pojačanom kroničnom upalom dišnih putova i pluća izazvanom stranim česticama i plinovima koja dovodi do stvaranja ispljuvka i razaranja plućnog parenhima. TLR receptori uključeni su u regulaciju urođenog i stečenog imuniteta te imaju ključnu ulogu u održavanju homeostaze pluća. Važni su u obrani od patogena, ali ne dozvoljavaju razvijanje pretjeranog upalnog odgovora koji bi mogao dovesti do oštećenja plućnog tkiva. Smatra se da cigaretni dim i drugi štetni čimbenici, u kombinaciji s genskom predispozicijom, djeluju na TLR receptore, dovode do njihove aktivacije, nakupljanja stanica imunosnog sustava u većem broju u dišnim putovima, otpuštanja upalnih citokina te promjena u ekspresiji gena za TLR receptore što pridonosi razvoju kronične upale. U ovom istraživanju ispitivala se toksičnost različitih koncentracija agonista TLR receptora LPS-a (agonist TLR4) i LTA (agonist TLR2) na NCI-H292 epitelnim stanicama traheo-bronhijalnog trakta. Na taj se način pokušao oponašati upalni odgovor u bolesnika s KOPB-om. Vijabilnost stanica određivala se tripanskim plavilom, određivanjem katalitičke aktivnosti LDH i MTS testom. Dobiveni rezultati su pokazali da 0,5 μg/mL LPS-a i 0,5 i 1 μg/mL LTA uzrokuju gubitak integriteta stanične membrane budući da je izmjerena povišena aktivnosti LDH u mediju iznad tretiranih stanica u odnosu na ukupan LDH u NCI-H292 stanicama. Međutim, MTS test je pokazao da te iste koncentracije ne dovode do značajne promjene redukcijske sposobnosti stanica u odnosu na kontrolu. Također, istraživanje je pokazalo da niska koncentracija LPS-a, 0,1 μg/mL, blago potiče proliferaciju NCI-H292 stanica u odnosu na kontrolu. Stoga smatramo da LPS i LTA u primijenjenim koncentracijama nisu značajno toksični za NCI-H292 stanice.

Abstract (english)

Chronic obstructive pulmonary disease is characterized by enhanced chronic airway and lung inflammation caused by inhalation of particles and gases which leads to sputum production and destruction of lung parenchyma. TLR receptors are involved in regulation of innate and acquired immunity and have a key role in maintenance of lung homeostasis. They play important part in defending against infections, but prevent excessive inflammatory response which could lead to lung destruction. It is believed that cigarette smoke and other risk factors, combined with genetics, affect TLR receptors, activate them and lead to enhanced recruitment of immune cells in the airway, proinflammatory cytokine production and changes in TLR receptor gene expression which contributes to development of chronic inflammation.
In this research we investigated toxicity of different concentrations of TLR receptor agonists LPS (TLR4 agonist) and LTA (TLR2 agonist) on NCI-H292 epithelial cells of tracheobronchial tract by mimicking inflammatory responses in COPD patients. Cell viability was determined with Trypan blue, LDH and MTT assays. According to the results 0.5 μg/mL LPS and 0.5 and 1 μg/mL LTA caused significant loss of cell membrane integrity as shown by increased LDH activity in surrounding medium of treated NCI-H292 cells in comparison with total LDH in the cells. On the other hand, MTT assay showed no significant changes in metabolic activity between treated cells and non-treated control cells. It was also determined that low doses of LPS (0.1 μg/mL) stimulate NCI-H292 cell proliferation as compared to non-treated control cells. Therefore we suggest that applied concentrations of LPS and LTA are not considerably toxic to NCI-H292 cells.

• Keywords: lipopolisaharid; lipoteična kiselina; TLR receptori; kronična opstrukcijska plućna bolest; NCI-H292 stanice
• Keywords (english): lipopolysaccharide; lipotheicoic acid; TLR receptors; chronic obstructive pulmonary disease; NCI-H292 cells
• Language: croatian
• URN:NBN: urn:nbn:hr:163:639295
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-01-20 11:00:28