İndüklenmiş Mezenşimal Kök Hücrelerden Elde Edilen Eksozomların in Vitro Yara Modelinde İyileşme Üzerine Etkisinin İncelenmesi

Abstract

The presented thesis discusses the healing potentials of UV light-induced and uninduced adipose and bone marrow-derived mesenchymal stem cell (MSC) exosomes in two- and three-dimensional wound models. For this purpose, different doses of UV-B (10, 25, 50, 100, and 200 mJ/cm2) were applied to MSCs originating from adipose tissue (AD) and bone marrow (BM). MTT method was used for cell viability analysis and Acridine orange / Propidium iodide dual staining method was used to show cell death. As a result of these analyses, it was determined that the most suitable non-toxic UV-B dose on cells was 25 mJ/cm2. Exosome isolations were made from MSCs to which the selected UV-B dose was applied or not. The exosomes obtained were characterized by various methods, and then ELISA analyses were performed to determine the growth factors and cytokine (IL-6, PDGF-BB, VEGF-A, TGF-Ꞵ) levels they contain. Firstly, human keratinocyte (HaCaT) and human fibroblast (HDF) cell lines were amplified to create in vitro two- and three-dimensional wound models. After co-culturing the fibroblast and keratinocyte cells to create a two-dimensional wound model, a 'scratch wound model' was created and different doses (1, 5, and 10 µg/ml) of the obtained exosomes were applied to the wound model. For cell proliferation monitoring, Alamar Blue analysis at 12, 24, 36, and 48 hours, and for wound closure analysis, measurements were made on photographs taken with an inverted microscope using the Image J program. According to the results of the scratch assay, it was concluded that the most effective dose in wound healing analyses from ADMSC exosomes was 10 µg/ml, and 5 µg/ml in BMMSC exosomes. Finally, for the three-dimensional wound model, first, the skin model and then the wounds were created with the 'Air-Liquid Interface' method in the transwell system. The exosome doses applied to the three-dimensional wound models were the doses obtained from the scratch wound model results. In this model, recovery rates were obtained from measurements taken randomly with Image J from histological sections to examine the healing stages. The results were found to support the two-dimensional model. In particular, the highest wound closure was observed at 10 µg/ml of UV-induced ADMSC exosomes, while the negative control group had the lowest wound closure rate. In conclusion, it was observed that UV-B-induced MSC exosomes have increased potential for healing in both two-dimensional and three-dimensional in vitro wound models. In order to examine the effects of UV light on MSC exosomes in more detail, molecular level analyses and in vivo experiments to examine its effect on wound healing will shed light on the studies in this field.