• Login
    View Item 
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    Transplantation of genetically modified hepatocytes containing a copper-transporting ATPase, direct hepatic delivery, and the potential for the treatment of Wilson's disease

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Find Full text
    Author
    Sanchez, Rigoberto A.
    Advisor
    DeTolla, Louis J.
    Date
    2005
    Type
    dissertation
    
    Metadata
    Show full item record
    Abstract
    Wilson's disease is caused by a single gene defect in a copper transporting ATPase. The ATP7b protein, expressed primarily in the liver, is non-functional in patients due to a mutation in the gene sequence. Patients with Wilson's disease typically present with increased copper concentrations, chronic hepatitis, and fulminant liver failure. The murine Wilson's Disease animal model is the ATP7b<tx-J> mouse strain. If viable hepatocytes can be extracted from donor rodents, and then be found in recipient rodents after intrasplenic injection, then the introduction of a cDNA encoding a functional ATP7b protein could perhaps help lower copper concentrations in recipient rodents afflicted with hepatic copper accumulation. Primary hepatocytes were isolated using retrograde perfusion, labeled, injected into recipient spleens, followed over a period of time, and then identified within the recipient liver and spleen. Preliminary observations suggested that intrasplenic transplantation of hepatocytes cause a consistent series of pathophysiological events, and can be found in the liver and spleen for several weeks. The RS-VPCR control vector was created by placing a control 500 base pair insert within the pcDNA 4/HisMax-TOPO plasmid. Numerous ratios of lipid to plasmid were tested to determine the highest rate of transfection. Recipient mice received an intrasplenic injection of syngeneic derived donor hepatocytes, transfected with the control insert sequence plasmid. PCR confirmed the presence of the control DNA sequence in the liver and spleen of the recipient animals at 1 and 2 days post injection. The ATPase gene was subcloned into a pcDNA 4/HisMax-TOPO plasmid and then transfected into hepatocytes, for intrasplenic transplant. Samples for all time points were negative for the ATP7b insert by PCR. As an alternate route, direct hepatic injection was conducted using cationic lipid and the ATP7b plasmid. At 7, 14, 21, and 28 days post direct hepatic injection, livers and spleens were collected for DNA, RNA and protein isolation, and blood was collected for serum copper analysis. DNA, RNA and proteins specific for the ATP7b insert were predominantly found in all samples for all time points. However, copper concentrations did not show a large significant change. Additional studies with enhanced protein expression could provide more information to allow improvement in copper concentrations in this Wilson's disease animal model.
    Description
    University of Maryland, Baltimore. Ph.D. 2005
    Keyword
    Biology, Molecular
    Biology, Cell
    Health Sciences, Medicine and Surgery
    ATP7b
    Hepatocytes--transplantation
    Hepatolenticular Degeneration--genetics
    Mice
    Models, Animal
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1095
    Collections
    Theses and Dissertations School of Medicine
    Theses and Dissertations All Schools

    entitlement

     
    DSpace software (copyright © 2002 - 2023)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.