Utjecaj utišavanja Toll sličnog receptora 9 na poticanje urođenog imunosnog odgovora nakon infekcije s adenoviralnim vektorom 26

master's thesis

• Title (english): Effect of silencing Toll like receptor 9 to promote innate immune response after infection with adenoviral vector 26
• Author: Marijana Slijepčević
• Mentor: Andreja Ambriović Ristov (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biology and Microbial Genetics); Zagreb
• Defense date and country: 2018-07-23, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Genska terapija označava unos genskog materijala u ciljanu stanicu u svrhu terapeutskog učinka, kako bi se popravio određeni poremećaj ili unijela neka nova funkcija u stanicu. Najistraživaniji adenovirus tipa 5 (AdV5) se najčešće koristi kao vektor u genskoj terapiji, no visoka seroprevalencija AdV5 predstavlja problem prilikom uporabe tog tipa vektora. Moguće rješenje ovog problema je korištenje adenovirusa sa niskom seroprevalencijom u populaciji kao što je adenovirus tipa 26 (AdV26). U ovom radu istražena je uloga Toll sličnog receptora 9 (TLR9) u poticanju urođenog imunosnog odgovora u epitelnim stanicama nakon infekcije AdV26. U stanicama adenokarcinoma pluća čovjeka (A549), korištenjem specifične siRNA (small interfering RNA), utišan je TLR9 te su korištenjem metoda Real Time RT-PCR i ELISA određene ekspresije gena i proteina koji sudjeluju u urođenom imunosnom odgovoru nakon infekcije AdV26. Dobiveni rezultati ukazuju da AdV26 nakon ulaska u stanice A549 potiče urođeni imunosni odgovor. Nakon infekcije s AdV26, dobivena je povećana ekspresija gena koji kodiraju za IL-6, IL-8, IL-18, IL-1β te IFN-β, a smanjena ekspresija gena koji kodira za IFN-α1. U stanicama sa smanjenom ekspresijom TLR9 potaknut imunosni odgovor bio je manji za većinu promatranih gena, u odnosu na stanice u kojima TLR9 nije bio utišan. Mogući mehanizam poticanja imunosnog odgovora nakon infekcije AdV26 ukjučuje P-ERK i P-p38 molekule. Također je uočeno da AdV26 bolje inficira stanice sa utišanim TLR9, što do sad nije bilo poznato.

Abstract (english)

Gene therapy refers to the entry of foreign gene material into the target cell for the purpose of therapeutic effect, in order to correct a particular disorder, or introduce a new function into the cell. The most investigated so far is adenovirus type 5 (AdV5) which is commonly used as a vector in gene therapy. High seroprevalence of AdV5 is high in the population causing a problem when using this type of vector. The possible solution is to use adenovirus whose seroprevalence is low in the population, as adenovirus type 26 (AdV26). The aim of this study is to investigate the role of Toll like receptor 9 (TLR9) in stimulating the innate immune response after AdV26 infection in epithelial cells. In human lung adenocarcinoma cells (A549), using specific interfering RNA (siRNA), TLR9 was suppressed and the level of mRNA and proteins expressed as innate immune response to AdV26 was investigated by the Real Time RT-PCR and ELISA methods. The obtained results indicate that AdV26 after entering the A549 cells promotes the innate immune response. After infection with AdV26, there was obtained increased expression of genes encoding for IL-6, IL-8, IL-18, IL-1β and IFN-β, and decreased expression of the gene encoding for IFN-α1. In cells with silenced TLR9 there was reduced immune response, the response was lower for most of the investigated genes compared to cells where TLR9 was not suppressed. Possible mechanism of promoting innate immune response after AdV26 infection includes P-ERK and P-p38. Moreover, the results show that AdV26 is better transduced in cells with silenced TLR9, which so far has not been reported.

• Keywords: adenovirus tipa 26; toll slični receptor 9; urođeni imunosni odgovor
• Keywords (english): Adenovirus type 26; toll like receptor 9; innate immune response
• Language: croatian
• URN:NBN: urn:nbn:hr:159:969695
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-12-02 13:36:02