Vpliv imunostimulacije primarne celične kulture epitelnih celic mlečne žleze goveda na izražanje provnetnih citokinov

Dular, Tea (author), Ogorevc, Jernej (mentor)

Mastitis oz. vnetje mlečne žleze je ena od gospodarsko najpomembnejših bolezni prežvekovalcev za prirejo mleka. Mastitis običajno povzročajo bakterije. Celične kulture lahko služijo kot alternativa študijam na živih živali in predstavljajo in vitro model za preučevanje poteka bolezni, kar je ključno za vzpostavitev uspešnih strategij za preprečevanje mastitisa. V eksperimentalnem delu diplomske naloge smo vzpostavili primarno celično kulturo epitelnih celic mlečne žleze goveda – pbMEC in jo uporabili za preučevanje imunskega odziva ob imunostimulaciji. pbMEC smo vzpostavili na neinvaziven način iz mleka in z imunofluorescenco potrdili prisotnost epitelnih celic. Celice smo imunostimulirali z virulentnim dejavnikom Gram negativnih bakterij LPS ter jih inkubirali 3 in 24 ur. Po pretekli inkubaciji smo izolirali RNA, jo reverzno prepisali v cDNA in z metodo qPCR določili diferenčno izražanje genov za citokina IL-8 in TNF-alfa. Rezultati so pokazali, da se je izražanje IL-8 in TNF-alfa po imunostimulaciji z LPS povečalo, kar kaže na to, da je kultura pbMEC imunokompetentna. Izražanje IL-8 je bilo po 24 urah višje kot po 3 urah, medtem ko je bilo izražanje TNF-alfa višje po 3 urah in je bilo po 24 urah le še nekoliko povečano. Rezultati so skladni z ugotovitvami prejšnjih študij ter kažejo na to, da je kultura pbMEC potencialno uporaben in vitro model za preučevanje imunskega odziva ob mastitisu.

• Language: Slovenian
• Keywords: pbMEC, mastitis, celični modeli, LPS, Escherichia coli, citokini, IL-8, TNF-alfa, qPCR
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2023
• PID: 20.500.12556/RUL-147083
• COBISS.SI-ID: 157727235
• Publication date in RUL: 23.06.2023

Secondary language

• Language: English
• Title: The effect of immunostimulation of primary cell culture of bovine mammary gland epithelial cells on the expression of proinflammatory cytokines

Abstract

Mastitis, inflammation of the mammary gland, is one of the most economically important diseases of dairy ruminants. Mastitis is usually caused by bacteria. Cell cultures can serve as an alternative to live animal studies and provide an in vitro model to study the course of the disease, which is crucial for the establishment of successful mastitis prevention strategies. In the experimental part of the thesis, we established a primary cell culture of bovine mammary epithelial cells – pbMEC, and used it as a cell model to study mastitis. pbMEC were established in a non-invasive manner from milk, and the presence of epithelial cells was confirmed by immunofluorescence. The cells were immunostimulated with the Gram negative bacterial virulence factor LPS and incubated for 3 and 24 hours. After the incubation, RNA was isolated, reverse transcribed into cDNA and the differential expression of the IL-8 and TNF-alpha cytokines was determined by qPCR. The results showed that the expression of IL-8 and TNF-alpha increased after immunostimulation with LPS, suggesting that the pbMEC culture is immunocompetent. The expression of IL-8 was higher after 24 hours than after 3 hours, while the expression of TNF-alpha was higher after 3 hours and was only slightly elevated after 24 hours. The results are consistent with the findings of previous studies and suggest that pbMEC culture is a potentially useful in vitro model to study the immune response during mastitis.

• Keywords: pbMEC, mastitis, cell models, LPS, Escherichia coli, cytokines, IL-8, TNF-alpha, qPCR