| dc.contributor.advisor | Siggelkow, Heide Prof. Dr. | |
| dc.contributor.author | Beismann, Johannes | |
| dc.date.accessioned | 2015-05-18T09:14:33Z | |
| dc.date.available | 2015-06-03T22:50:12Z | |
| dc.date.issued | 2015-05-18 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0022-5FE4-7 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-5071 | |
| dc.language.iso | deu | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | |
| dc.subject.ddc | 610 | de |
| dc.title | Der Einfluss der Überexpression des Enzyms 11-β Hydroxysteroid-Dehydrogenase Typ 1 auf die adipogene Differenzierung von mesenchymalen Vorläuferzellen in vitro | de |
| dc.type | doctoralThesis | de |
| dc.title.translated | The impact of 11-β-hydroxysteroid dehydrogenase type 1 on adipogenic differentiation in mesenchymal progenitor cells | de |
| dc.contributor.referee | Miosge, Nicolai Prof. Dr. | |
| dc.date.examination | 2015-05-27 | |
| dc.description.abstracteng | Introduction: The differentiation of mesenchymal progenitor cells (MSCs) to the osteoblastic or adipogenic phenotype is thought to be an important pathophysiological mechanism for bone diseases. Dexamethasone induced adipogenesis in mesenchymal progenitor cells by decreasing osteogenic differentiation. The internal cortisol metabolism in bone seems to be mainly regulated by the enzyme 11-β HSD1, a key enzyme converting cortisone to active cortisol or vice versa. We examined overexpression of 11- β HSD1 in relation to adipogenic transcription markers and the product cortisol. Cortisol has a widespread effect on bone metabolism. It suppresses the differentiation of the osteoblastic mesenchymal stem cells via RUNX and influences them in a proadipogenic way. An important regulator for this pathway are the early proadipogenic markers PPARγ, C/EBP alpha and beta. This project aims to promote the aspects of 11-β HSD1 overexpression in mesenchymal stem cells. This knowledge might help us understand the pathogenesis of aged-related osteoporosis. Material and methods: Mesenchymal progenitor cells from different patients were cultured in DMEM 10% FCS, glutamine, penicillin/streptomycin. mRNA was analyzed by SYBR Green® PCR. 11-β HSD1 and 11-β HSD2-protein were analyzed by western blotting. Cortisol and cortisone synthesis was measured by mass spectrometry. Mesenchymal progenitor cells were seeded 100.000/ ml in 6 well plates and transfected for 6 h at confluence using lipofectamine and a pcDNA3.1 plasmid for 11-β HSD1 overexpression (provided by V. Bähr) or control plasmid. Mesenchymal progenitor cells after transfection were cultured for either 48 hours, 7 days or up to 28 days. Amongst others we used a stimulation medium to provide enzymatic activity and to generate enzymatic cofactors. The added factors were cortisone and cortisol. Furthermore we added NADPH, NAD, glucose-6-phosphate and glucose-6-phosphate-dehydrogenase, all together these factors arise a regeneration system for 11-β HSD1 cofactors. Results: After addition of cortisone for 48h cortisol synthesis doubled after transfektion with 11-β HSD1 in cell suspension and media. By comparison, after addition of cortisol for 48 hours cortisone synthesis did not increase in cell suspension and not very distinctive in media. This result accorded with a spontaneous conversion of cortisol in cortisone. We transfected 11-β HSD1 in mesenchymal progenitor cells, expression increased 10 to 1000 fold depending on the individual experiment. 11-β HSD1, assisted by cofactors (regeneration system), increased synthesis of cortisol more than without cofactors. mRNA Expression remained stable over 7 days and decreased thereafter. Plasmid transfection increased 11-β HSD1 protein detectable by western blotting after 48 hours and 28 days. In addition 11-β HSD2 protein also increased after 48h. Even so the adipogenic differentiation, measured by mRNA of aP2, C/EBP alpha and C/EPB beta was clearly induced after 48 hours and 7 days. The osteogenic differentiation measured by mRNA of RUNX2 was significantly suppressed after 28 days. mRNA of RANKL was induced after 48 hours as well. Conclusions: In our experimental setup we show that a 11-β HSD1 overexpression is in mRNA and protein levels possible and repeatable. Our results suggest an increased proadipogenic differentiation of mesenchymal progenitor cells by overexpression without stimulation. Furthermore, we could show the direct detection of the increased product cortisol and the influence of cofactors on the enzyme 11-β HSD1. The implantation of a regenerations system is usefully and supports the thesis of an increased conversion of cortisone in cortisol in mesenchymal progenitor cells, like it might appear in age-related osteoporosis. | de |
| dc.contributor.coReferee | Schön, Margarete Prof. Dr. | |
| dc.subject.eng | bone | de |
| dc.subject.eng | cortisol | de |
| dc.subject.eng | age related osteoporosis | de |
| dc.subject.eng | transfection | de |
| dc.subject.eng | plasmid | de |
| dc.subject.eng | 11-β HSD1 | de |
| dc.subject.eng | MSCs | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0022-5FE4-7-3 | |
| dc.affiliation.institute | Medizinische Fakultät | de |
| dc.subject.gokfull | Medizin (PPN619874732) | de |
| dc.description.embargoed | 2015-06-03 | |
| dc.identifier.ppn | 82557840X | |