The effect of applied paediatric burn blister fluid and extracted exosomes from different depths on human dermal fibroblasts and keratinocytes
Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2019
Författare
Weinefors, Erika
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Burn injuries are common types of wounds which have different degrees of severity depending on the affected tissue depth and size. The depths are classified as superficial-partial thickness, deep-partial thickness or full thickness. During the burn wound healing process fluid filled blisters can form in the skin, however the effect of these are not fully understood yet. This Master’s thesis project aimed to examined the effect of paediatric burn blister fluid and isolated exosomes on human foreskin fibroblasts (HFFs) and partly on keratinocyte-like HaCaTs. Assays such as WST-1, TGF-β pathway real-time PCR, enzyme-linked immunosorbent (ELISA) and microscopic methods were used to examine the metabolic and proliferative activity as well as gene expression and cytokine production. The exosomes were extracted from burn blister fluid using ultracentrifugation and characterized using western blot, Transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). The project have successfully been able to, for the first time, characterize exosomes in burn blister fluid from different burn depths. The exosomes were detectable in TEM images and results obtained from the NTA showed particles predominantly in the size 150 nm in the concentration 1010-11 particles/ml. Exosomes did not have a significant proliferative effect neither increased the metabolic activity of treated HFFs cultures. Real-time PCR and ELISA results showed an gene up-regulation of TGF-β1 at 24 h and at 48 h its physical presence in deep exosome treated HFF cultures. The result showed a down-regulation of COL1A1 and IL-6 at 24 and 48 h in superficial and full thickness exosome cultures and no sign of cytokine production. Whole burn blister fluid from superficial-partial and deep-partial paediatric burns were used in the same functional assays. The obtained result showed that burn blister fluid have the ability to increase the metabolic activity and proliferation of HFFs as well as HaCaTs, altough not in the same extent. At 24 h burn blister treated HFFs have an up-regulated gene expression of TGF-β1 and a downregulated expression of COL1A1 and IL-6 compared to serum free control, this is however not replicated at the same timepoint in the ELISA result, were no TGF-β1 was detected. IL-6 and IL-1β was detected in almost all burn blister sample treated HFF cultures. The result indicates that burn blister fluid and extracted exosomes have various effects on HFFs and HaCats.
Beskrivning
Ämne/nyckelord
Biokemi och molekylärbiologi , Mikrobiologi , Medicinsk bioteknik , Livsvetenskaper , Biochemistry and Molecular Biology , Microbiology , Medical Biotechnology , Life Science