Vpliv bakterije Mycoplasma canis na izraženje vnetnih genov na modelu trajne celične linije DH82

Glinšek, Katja (author), Narat, Mojca (mentor), Starčič Erjavec, Marjanca (reviewer)

Mycoplasma canis je bakterija, oportunistični patogen, ki je bila izolirana iz psov z urogenitalnimi boleznimi in težavami s plodnostjo. Izolirali so jo tudi iz tkiv drugih sesalcev. Eksperimentalne okužbe z M. canis povzročajo kronična vnetja sečnice in obmodka ter vnetje stene maternice. Okužbo z M. canis povezujejo tudi z neplodnostjo pri psih, vendar direktnega dokaza za to še ni. O pasjih mikoplazmah in njihovih viruletnih dejavnikih, patogenosti ter učinkih na gostitelja ni veliko podatkov. V okviru magistrske naloge smo preverili vpliv okužbe trajne pasje celične linije DH82 z M. canis. V določenih časovnih intervalih smo opazovali vpliv na viabilnost celic DH82 in njihovo morfologijo ter z metodo PCR v realnem času spremljali izražanje genov za vnetne citokine IL-1β, IL-6, IL-8 in transkripcijski dejavnik NFĸB1. Merili smo tudi izločanje dušikovega oksida (NO) in število živih bakterij v supernatantih celic DH82. Ugotovili smo, da se je izražanje gena za IL-1β tekom okužbe zviševalo. Izražanje gena za IL-6 se je zviševalo do 6 h po okužbi, medtem ko je po 12 h začelo padati. Izražanje gena za IL-8 se je tekom okužbe postopno poviševalo in je bilo najvišje po 48 h. Vpliv na viabilnost celic DH82 se je začel kazati 24 h po okužbi in je bil po 48 h še bolj izrazit. Koncentracija živih bakterij v supernatantih celic DH82 se je tekom okužbe spreminjala in je bila najnižja 48 h po okužbi. Spremenjenih koncentracij NO v supernatantih DH82 nismo zaznali. S pridobljenimi rezultati smo dobili delno sliko poteka okužbe evkariontskih celic z M. canis.

• Language: Slovenian
• Keywords: mikoplazme/Mycoplasma canis/vnetje/okužba/citokini/izražanje genov/IL-1/IL-6/IL-8/NF&#954, B/DH82
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2017
• PID: 20.500.12556/RUL-95597
• COBISS.SI-ID: 4477007
• Publication date in RUL: 21.09.2017

Secondary language

• Language: English
• Title: INFLUENCE OF Mycoplasma canis INFECTION ON THE EXPRESSION OF INFLAMMATORY RESPONSE GENES IN CANINE MACROPHAGE CELL LINE DH82

Abstract

Mycoplasma canis is an opportunistic pathogen of dogs, isolated from dogs with urogenital diseases and infertility. It has also been isolated from other mammals. Experimental infections with M. canis produced chronic urethritis, epididymitis and endometritis. However, more work is needed to establish whether M. canis is linked to infertility. Little is known about virulence factors and pathogenicity of canine mycoplasmas and their effect on the host. We analyzed the influence of M. canis infection on the immortilized canine cell line DH82. After infection of the DH82 cell line with M. canis we followed the changes in viability and morphology of DH82 cells. Using the real-time PCR method, we analyzed relative changes in gene expression of inflammatory cytokines IL-1β, IL-6, IL-8 and transcription factor NFĸB1. Moreover, we analyzed the release of nitric oxide (NO) and the concentration of viable bacteria in DH82 cell supenatants during infection. The infection resulted in gradual increase of IL-1β gene expression. The expression of IL-6 gene gradually increased until 6 h after infection and dropped until 12 h after infection. Also IL-8 gene expression gradually increased during infection, however the expression was the highest 48 h after the infection. The viability of DH82 cells dropped 24 h and even more obviously 48 h after infection. The concentration of viable bacteria in DH82 cell supernatants varied during the infection and it was the lowest 48 h after the infection. We did not detect any changes in NO concentrations in DH82 cell supernatants after the infection. The gained results give us a partial insight into the M. canis infection of the eukaryotic cells.

• Keywords: mycoplasmas/Mycoplasma canis/inflammation/infection/cytokines/gene expression/IL-1/IL-6/IL-8/NF&#954, B/DH82