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Adhesion and growth of adipose tissue-derived stem cells on fibrin assemblies with attached growth factors for tissue engineering of heart valves
Filová, Elena ; Trávníčková, Martina ; Knitlová, Jarmila ; Matějka, Roman ; Kučerová, Johanka ; Riedelová, Zuzana ; Brynda, Eduard ; Bačáková, Lucie
Currently used xenogeneic biological heart valve prostheses are decellularized and crosslinked with glutaraldehyde. These grafts usually undergo degeneration and calcification. Pericardium-based heart valve prostheses, re-seeded with autologous cells, i.e. Adipose tissue-derived cells (ASCs) and endothelial cells, could have longer durability and biocompatibility. In order to improve the adhesion of cells and their ingrowth into decellularized pericardium, various fibrin (Fb) layers were developed, i.e. Fb, Fb with covalently bound heparin (H), Fb with either vascular endothelial growth factor (VEGF) or fibroblast growth factor 2 (FGF) in various concentrations (1 ng/ml, 10 ng/ml, 100 ng/ml) or with both VEGF and FGF (100 ng/ml). Growth factors were attached onto Fb via heparin or were adsorbed. ASCs were seeded on theses layers in a DMEM medium supplemented with 2 % of fetal bovine serum, TGFβ1 and BMP-4 (both 2.5 ng/ml), and with ascorbic acid. Cell adhesion and growth/viability was assessed by counted cell number/MTS evaluation. ASCs were stained for differentiation markers of smooth muscle cells, such as alpha-actin, calponin, and myosin heavy chain. On day 7, ASCs on Fb-H-VEGF layers produced both calponin and alpha-actin. An increased FGF concentration caused reduced calponin staining of ASCs. Lack of heparin in fibrin assemblies with growth factors inhibited the production of both alpha-actin and calponin in ASCs. The highest ASCs density/viability was found on Fb-H-VEGF-FGF layer. The proper formulation of fibrin coatings could be favorable for ASCs growth and differentiation and could subsequently support endothelialization of cardiovascular prostheses with endothelial cells.
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Role of expression of cellular prion protein in the differentiation of neuronal cell lines
Kučerová, Johanka ; Holada, Karel (advisor) ; Janoušková, Olga (referee)
Cellular prion protein (PrPC ) is a membrane bound glycoprotein. The protein is expressed in all vertebrates, mainly in the nervous system, but it is present also in the cells of gastrointestinal tract, bone marrow, germ cells and heart. PrPC is necessary for pathogenesis of prion diseases, which are deadly and without the possibility of therapy. The pathogenic isoform of prion protein is formed by changing of secondary structure of PrPC and it's the main constituent of infectious prion particles. Pathological form of prion protein accumulates in brain of infected patients and this process is associated with neurodegradation. Physiological function of PrPC is poorly understood. Knock-out of the PrPC gene (PRNP) is not connected with any noticeable phenotype. Potential functions of PrPC are dispersed, protein may have antiapoptotic effect, it can be involved in ions metabolism or in protection against oxidative stress. Latest results show, that PrPC can play important role in cell differentiation. During the differentiation PrPC can influence the development of cells and their typing. It could affect cell cycle and have an influence on formation of nervous system. Aim of the present study was to elucidate, whether the down-regulation of PrPC or infection with prions has an impact on differentiation of...
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Role of prion proteins at differentiating cells
Kučerová, Johanka ; Holada, Karel (advisor) ; Drbal, Karel (referee)
Cellular prion protein (PrPC) is well known for its pathological isoform PrPSc, widely believed to be the infectious agent of the prion diseases, which include Bovine spongiform encephalopathy (BSE), scrapie and Creutzfeldt-Jakob disease. The physiological role of PrPC is poorly understood, but its involvement in the regulation of apoptosis, adhesion molecules, antioxidant, or signal molecules, has been described. Despite of these findings, it hasn't been proven, that the protein is necessary for normal development of mice. However, the protein was shown to be essential for regeneration of hematopoietic stem cells after exposure to lethal stress conditions. Expression of PrPC may have an effect on the proliferation and differentiation of cells by helping them keep the proliferative activity, or slow spontaneous differentiation. The quantity of the protein correlates positively or negatively with expression of transcription factors such as Oct4/Nestin, which are essential for development in embryogenesis. Its expression also regulates transition of cells from G1 phase to S phase of the cell cycle. This bachelor thesis is focused on published results describing the influence of PrPC on cellular proliferation and differentiation.
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