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Differentiation of Human Embryonic Stem Cells into Endothelial and Smooth Muscle Cells as a Model for Vascular Development
Obrtlíková, Petra ; Trněný, Marek (advisor) ; Mokrý, Jaroslav (referee) ; Hampl, Aleš (referee)
Aims of the study: We hypothesized that the optimal source of cell for vascular regeneration will be the progenitor cells derived from human embryonic stem cells (ESCs) which can differentiate both into endothelial cells (ECs) as well as vascular smooth muscle cells (SMCs). We propose to test if the population of human ESCs, H9 cell line, can serve this role. Material and methods: Human ESCs were cocultured with stromal cells S17, M2-10B4 or Wnt1 expressing M2-10B4 cell line to generate a CD34+ cell population. After that, CD34+ cells were sorted and cultured in media containing specific cytokines to generate ECs. To induce SMC differentiation from ECs, culture conditions were changed to media containing platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-b1). Phenotypic and functional characteristics of these populations were demonstrated by flow cytometry, immunohistochemistry, QRT- PCR, tube formation assay, and response to calcium signaling agonists. Results: CD34+ vascular progenitor cells derived from human ESCs give rise to ECs and SMCs. These two populations express cell specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Wnt1 overexpressing stromal cells...
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Genetic factors in lymphoproliferative malignancies Focus on CHEK2 gene in lymphomas with comparison to distinct solid tumors
Havránek, Ondřej ; Trněný, Marek (advisor) ; Papajik, Tomáš (referee) ; Veselý, Pavel (referee)
Genetic factors in lymphoproliferative malignancies. Focus on CHEK2 gene in lymphomas with comparison to distinct solid tumors. MUDr. Ondřej Havránek Summary of PhD thesis: Background: The checkpoint kinase 2 gene (CHEK2) codes for an important mediator of DNA damage response pathway that among others interacts with the p53 protein. Mutations in the CHEK2 gene increase the risk of several cancer types, however, their role in non- Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) is not clear. The most frequent TP53 gene R72P polymorphism was analyzed in several studies in NHL but not in HL. Methods: We have performed mutation analysis of the whole CHEK2 gene coding sequence in 340 NHL patients and the segment coding for CHEK2 forkhead-associated (FHA) domain in 298 HL patients and compared the results with our analyses of CHEK2 in breast, colorectal and pancreatic cancers. The TP53 R75P genotype was assessed in the same lymphoma populations. Both genes were analyzed using denaturing high-performance liquid chromatography. Results: The overall frequency of CHEK2 alterations within FHA-coding region was significantly higher in NHL and HL patients (19/340 - 5.6%; 17/298 - 5.7%, respectively) compared to non-cancer controls (19/683 - 2.8%; p = 0.03 and 0.04, respectively). These alterations were associated with...
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Distinguishing of primary mediastinal B-cell lymphoma and diffuse large B-cell lymphoma with real-time quantitative polymerase chain reaction
Votavová, Hana ; Trněný, Marek (advisor) ; Papajik, Tomáš (referee) ; Kozák, Tomáš (referee)
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. It is a molecular and prognostic heterogeneous disease. Three main genetic subtypes are called germinal center-like DLBCL (GC-like DLBCL), non-germinal center-like DLBCL (nonGC-like DLBCL) and primary mediastinal B-cell lymphoma (PMBL). These subtypes can be reliably distinguished only with usage of gene expression profiling (GEP). The GEP method can be applied only when fresh frozen tissue is available. The method is technically difficult and expensive. Thus, it is not used routinely. Since the DLBCL subtypes differ in prognosis, it is extremely important to be able to distinguish them. The presented thesis is focused on distinguishing of PMBL diagnosis in the group of DLBCL. Easily stored formalin-fixed, paraffin-embedded tissue (FFPE) and gene expression analysis using real-time quantitative polymerase chain reaction (RTqPCR) are used. In the first step, PMBL and DLBCL cases were distinguished with an internationally accepted clinical-pathological method. The agreement between clinical-pathological diagnosis and GEP is only 76%. In the presented text a genetic algorithm for PMBL/DLBCL distinguishing is suggested. It uses three carefully chosen genes and their expression is measured with RTqPCR. Both, the...
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Human multipotent mesenchymal stromal cells - bone differentiation and hematopoietic support
Pytlík, Robert ; Trněný, Marek (advisor) ; Filová, Elena (referee) ; Lesný, Petr (referee)
Human mesenchymal stromal cells (hMSC) are adult stem or progenitor cells, which physiological role is reparation of damaged tissues. This is achieved mostly by secretion of trophic, angiopoietic and immunomodulatory factors. Beside this, hMSC have potential to differentiate in vitro into specialized cells, especially of the mesodermal lineages. Human MSC also significantly support hematopoiesis in hematopoietic niche. This knowledge raised high hopes for therapeutic use of hMSC, especially in regenerative medicine and treatment of autoimmune diseases, including graft versus host disease (GvHD). As a proof of concept served initially crude preparations of bone marrow mononuclear cells (BMMC), which contain small numbers of hMSC. In our hospital, two pilot clinical studies with BMMC were performed: study of treatment of acute myocardial infarction (negative, prematurely terminated) and study of treatment of peripheral leg arthery disease (promising results). Further research was aimed on optimalisation of hMSC cultivation method for clinical use to obtain highest possible yield and get free from animal proteins. Human MSC were traditionally cultivated in research-grade media with fetal calf serum (FCS), which can lead to immunization of patients after repeated application of hMSC. We achieved...
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