{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:62272"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:62272","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Molekulares Targeting von Hepatischen Sternzellen zur Therapie der Leberfibrose","abstract":"Chronic injury of the liver leads to liver fibrosis by the activation of hepatic stellate cells (HSC) and in final consequence to liver cirrhosis. Removal of activated HSC by apoptosis precedes fibrosis resolution in experimental induced fibrosis. Thus the purpose of this work was to induce apoptosis by overexpression of pro-apoptotic proteins in activated HSC specifically. Therefore, the genes for the pro-apoptotic proteins p53, Bax and HSV1tk were cloned under the transcriptional control of a human CSRP2-promoter fragment. Those gene cassettes have been placed in replication deficient adenoviruses to infect HSC. In different assays it has been shown, that the cloned CSRP2-p53 construct could not induce a significant apoptosis in rat HSC. One possible reason for this could be the self-limitation of this construct, which was shown by different reporter gene assays in this thesis. An even more dramatic self-limitation was also shown for the CSRP2-bax construct. Furthermore, the comparison of homology and expression shows, that the human promoter fragment is highly differentially regulated and expressed in human HSC versus rat HSC. The second aim of this thesis was the analysis of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres with mannose 6 phosphate (M6P) ligands as a specific delivery-system for activated HSC in cooperation with the Department of Textile Chemistry and Macromolecular Chemistry of RWTH Aachen. It was shown, that in vivo systemically applicated nanospheres mainly accumulated in liver and spleen after 24 hours and did not cause lethal embolisms. Additionally performed analyses of the M6P/IGFII receptor expression in different organs however showed a higher expression of the receptor in heart and muscle cells than in the liver. Thus, the accumulation of the spheres in the liver seems not to be receptor mediated. Via confocal microscopy it was shown, that all types of liver cells incorporate the nanospheres in vitro, particularly the Kupffer cells. Yet there is no doubtless proof of the M6P ligand on the surface of the spheres, neither from the producer of the nanospheres nor in this thesis. Therefore, the observed effect seems to be an unspecific, non-receptor mediated incorporation in vitro, too. In conclusion, PLGA-nanospheres may serve as a liver specific delivery system in vivo, as they accumulate in liver after systemical application and are incorporated from every liver cell type in vitro. A HSC specific gene expression could be achieved with the studied human CSRP2 promoter fragment, but the results of different experiments with the p53 and bax genes under the transcriptional control of this promoter fragment showed a self-limitation of those constructs. However, a combination of this promoter fragment with the HSV1tk gene may lead to the intended specific induction of apoptosis in human HSC. This construct may therefore be a possible load for the PLGA-nanospheres for the specific targeting of activated HSC.","abstract_html":"Chronic injury of the liver leads to liver fibrosis by the activation of hepatic stellate cells (HSC) and in final consequence to liver cirrhosis. Removal of activated HSC by apoptosis precedes fibrosis resolution in experimental induced fibrosis. Thus the purpose of this work was to induce apoptosis by overexpression of pro-apoptotic proteins in activated HSC specifically. Therefore, the genes for the pro-apoptotic proteins p53, Bax and HSV1tk were cloned under the transcriptional control of a human CSRP2-promoter fragment. Those gene cassettes have been placed in replication deficient adenoviruses to infect HSC. In different assays it has been shown, that the cloned CSRP2-p53 construct could not induce a significant apoptosis in rat HSC. One possible reason for this could be the self-limitation of this construct, which was shown by different reporter gene assays in this thesis. An even more dramatic self-limitation was also shown for the CSRP2-bax construct. Furthermore, the comparison of homology and expression shows, that the human promoter fragment is highly differentially regulated and expressed in human HSC versus rat HSC. The second aim of this thesis was the analysis of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres with mannose 6 phosphate (M6P) ligands as a specific delivery-system for activated HSC in cooperation with the Department of Textile Chemistry and Macromolecular Chemistry of RWTH Aachen. It was shown, that in vivo systemically applicated nanospheres mainly accumulated in liver and spleen after 24 hours and did not cause lethal embolisms. Additionally performed analyses of the M6P/IGFII receptor expression in different organs however showed a higher expression of the receptor in heart and muscle cells than in the liver. Thus, the accumulation of the spheres in the liver seems not to be receptor mediated. Via confocal microscopy it was shown, that all types of liver cells incorporate the nanospheres in vitro, particularly the Kupffer cells. Yet there is no doubtless proof of the M6P ligand on the surface of the spheres, neither from the producer of the nanospheres nor in this thesis. Therefore, the observed effect seems to be an unspecific, non-receptor mediated incorporation in vitro, too. In conclusion, PLGA-nanospheres may serve as a liver specific delivery system in vivo, as they accumulate in liver after systemical application and are incorporated from every liver cell type in vitro. A HSC specific gene expression could be achieved with the studied human CSRP2 promoter fragment, but the results of different experiments with the p53 and bax genes under the transcriptional control of this promoter fragment showed a self-limitation of those constructs. However, a combination of this promoter fragment with the HSV1tk gene may lead to the intended specific induction of apoptosis in human HSC. 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Removal of activated HSC by apoptosis precedes fibrosis resolution in experimental induced fibrosis. Thus the purpose of this work was to induce apoptosis by overexpression of pro-apoptotic proteins in activated HSC specifically. Therefore, the genes for the pro-apoptotic proteins p53, Bax and HSV1tk were cloned under the transcriptional control of a human CSRP2-promoter fragment. Those gene cassettes have been placed in replication deficient adenoviruses to infect HSC. In different assays it has been shown, that the cloned CSRP2-p53 construct could not induce a significant apoptosis in rat HSC. One possible reason for this could be the self-limitation of this construct, which was shown by different reporter gene assays in this thesis. An even more dramatic self-limitation was also shown for the CSRP2-bax construct. Furthermore, the comparison of homology and expression shows, that the human promoter fragment is highly differentially regulated and expressed in human HSC versus rat HSC. The second aim of this thesis was the analysis of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres with mannose 6 phosphate (M6P) ligands as a specific delivery-system for activated HSC in cooperation with the Department of Textile Chemistry and Macromolecular Chemistry of RWTH Aachen. It was shown, that in vivo systemically applicated nanospheres mainly accumulated in liver and spleen after 24 hours and did not cause lethal embolisms. Additionally performed analyses of the M6P/IGFII receptor expression in different organs however showed a higher expression of the receptor in heart and muscle cells than in the liver. Thus, the accumulation of the spheres in the liver seems not to be receptor mediated. Via confocal microscopy it was shown, that all types of liver cells incorporate the nanospheres in vitro, particularly the Kupffer cells. Yet there is no doubtless proof of the M6P ligand on the surface of the spheres, neither from the producer of the nanospheres nor in this thesis. Therefore, the observed effect seems to be an unspecific, non-receptor mediated incorporation in vitro, too. In conclusion, PLGA-nanospheres may serve as a liver specific delivery system in vivo, as they accumulate in liver after systemical application and are incorporated from every liver cell type in vitro. A HSC specific gene expression could be achieved with the studied human CSRP2 promoter fragment, but the results of different experiments with the p53 and bax genes under the transcriptional control of this promoter fragment showed a self-limitation of those constructs. However, a combination of this promoter fragment with the HSV1tk gene may lead to the intended specific induction of apoptosis in human HSC. This construct may therefore be a possible load for the PLGA-nanospheres for the specific targeting of activated HSC."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University XIII, 166 S. : Ill., graph. Darst. (2007). = Aachen, Techn. Hochsch., Diss., 2007"]},{"key":"dc:title","label":"Title","values":["Molekulares Targeting von Hepatischen Sternzellen zur Therapie der Leberfibrose"]}]}],"canonical_facts":{"dc:contributor":["Peterhänsel, Christoph"],"dc:coverage":["DE"],"dc:creator":["Benner, Susanne"],"dc:date":["2007"],"dc:description":["Chronic injury of the liver leads to liver fibrosis by the activation of hepatic stellate cells (HSC) and in final consequence to liver cirrhosis. Removal of activated HSC by apoptosis precedes fibrosis resolution in experimental induced fibrosis. Thus the purpose of this work was to induce apoptosis by overexpression of pro-apoptotic proteins in activated HSC specifically. Therefore, the genes for the pro-apoptotic proteins p53, Bax and HSV1tk were cloned under the transcriptional control of a human CSRP2-promoter fragment. Those gene cassettes have been placed in replication deficient adenoviruses to infect HSC. In different assays it has been shown, that the cloned CSRP2-p53 construct could not induce a significant apoptosis in rat HSC. One possible reason for this could be the self-limitation of this construct, which was shown by different reporter gene assays in this thesis. An even more dramatic self-limitation was also shown for the CSRP2-bax construct. Furthermore, the comparison of homology and expression shows, that the human promoter fragment is highly differentially regulated and expressed in human HSC versus rat HSC. The second aim of this thesis was the analysis of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres with mannose 6 phosphate (M6P) ligands as a specific delivery-system for activated HSC in cooperation with the Department of Textile Chemistry and Macromolecular Chemistry of RWTH Aachen. It was shown, that in vivo systemically applicated nanospheres mainly accumulated in liver and spleen after 24 hours and did not cause lethal embolisms. Additionally performed analyses of the M6P/IGFII receptor expression in different organs however showed a higher expression of the receptor in heart and muscle cells than in the liver. Thus, the accumulation of the spheres in the liver seems not to be receptor mediated. Via confocal microscopy it was shown, that all types of liver cells incorporate the nanospheres in vitro, particularly the Kupffer cells. Yet there is no doubtless proof of the M6P ligand on the surface of the spheres, neither from the producer of the nanospheres nor in this thesis. Therefore, the observed effect seems to be an unspecific, non-receptor mediated incorporation in vitro, too. In conclusion, PLGA-nanospheres may serve as a liver specific delivery system in vivo, as they accumulate in liver after systemical application and are incorporated from every liver cell type in vitro. A HSC specific gene expression could be achieved with the studied human CSRP2 promoter fragment, but the results of different experiments with the p53 and bax genes under the transcriptional control of this promoter fragment showed a self-limitation of those constructs. However, a combination of this promoter fragment with the HSV1tk gene may lead to the intended specific induction of apoptosis in human HSC. This construct may therefore be a possible load for the PLGA-nanospheres for the specific targeting of activated HSC."],"dc:identifier":["https://publications.rwth-aachen.de/record/62272","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123849%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-19190"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University XIII, 166 S. : Ill., graph. Darst. (2007). = Aachen, Techn. 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