İnsan Göbek Kordon Veni CD146+ Hücrelerin ve Bu Hücrelerden Elde Edilen Matriks Proteinlerinin in vitro Biyofilm Modellerinde Değerlendirilmesi

Abstract

In the treatment of wounds, the application of cells or decellularized matrices with antimicrobial effects to the wound area has attracted attention in recent years. The aim of this thesis is to evaluate CD146+ stem cells and extracellular matrix proteins derived from these cells (decellularized matrix) which are thought to have antimicrobial effect in in vitro biofilm models. In this thesis, the characterization of CD146+ stem cells was evaluated by determination of cell surface markers and differentiation analyzes and characterization of decellularized matrix was done with histological/immunofluorescence staining and proteome analysis. To observe the contribution of CD146+ stem cells to biofilm formation, cell viability and proliferation were evaluated by WST-1, Annexin V/PI and crystal violet by co-culture with Pseudomonas (P.) aeruginosa and Staphylococcus (S.) aureus bacteria. Disc diffusion method was also used to observe possible antimicrobial effects of CD146+ stem cells. The effects of CD146+ stem cells and decellularized matrix proteins on biofilm formation were tested in a drip flow model and expression levels of the genes involved in biofilm formation were determined by quantitative reverse transcriptase-PCR method. As a result of the data obtained; it was determined that CD146/105+ stem cells could differentiate in adipogenic direction but they did not have osteogenic differentiation capacity. The characterization of the decellularized matrix was determined by DAPI and Masson’ s Trichrome staining, followed by proteome analysis to identify collagen, glycoproteins, proteoglycan and growth factors as matrix components. P. aeruginosa and S. aureus bacteria increased their proliferation capacity when they were cultured in stem cells and decellularized matrices. In the designed biofilm model by using P. aeruginosa and S. aureus, it was determined that the decellularized matrices significantly reduced the formation of P. aeruginosa biofilm, but there were no significant differences with S. aureus in the decellularized group compared to the cellular group. According to the results of gene expression analysis, significant decrease was found for rhlR and LasR among P. aeruginosa biofilm related genes that supports cell counting results. All of the icaADBC genes, which are the attachment genes of S. aureus, were highly expressed in the decellularized group. This thesis is the first study that demonstrates the importance of human stem cells and matrix proteins obtained from them, for clinical applications.