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Viral mediated gene transfer for modification of cell signalling and remodelling in the ischaemic myocardium
Başlık:
Viral mediated gene transfer for modification of cell signalling and remodelling in the ischaemic myocardium
Yazar:
Queen, Jillian May, author.
ISBN:
9780438060401
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (259 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Advisors: Howard Prentice.
Özet:
Myocardial ischaemia, the most common cause of mortality in developed societies, is characterised by contractile, electrical and biochemical alterations caused by a reduction of blood flow. Gene therapy is an increasingly attractive method of therapy for cardiovascular disease. The vector containing the potentially therapeutic gene must be able to target the cells within the myocardium specifically, have a high transduction efficiency, and the subsequent expression levels of the genes from such vectors must be capable of tight regulation. For the purpose of this study, two viral vectors were employed, the retrovirus and the adenovirus. The retroviral vector can specifically transduce mitotic cells, and was used for infection of isolated cardiac fibroblasts. The adenovirus was used to infect cardiac fibroblasts and cardiac myocytes, in vitro. The cardiac fibroblasts were shown to be effectively transduced by both vector systems. The cardiac myocytes were more successfully transduced by adenovirus. The successful transfer of foreign genes into the cardiac cells (fibroblasts and myocytes) was initially demonstrated using the LacZ gene encoding the bacterial cytoplasmic enzyme, beta-galactosidase. Enzyme activity was histochemically analysed by X-gal staining. The myocardium has no regenerative capacity and myocyte loss within an area of infarct will be permanent and will result in scar formation. This non-contracting scar is detrimental to cardiac function. Increasing the amount of viable muscle within this area of infarct should improve function. Analyses of the transfer of the skeletal muscle determination gene, MyoD, into isolated cardiac fibroblasts and also an area of infarct is discussed in chapter 5. In this study, the MyoD gene was transferred in an adenoviral and retroviral vector. Recombinant retrovirus and adenovirus vectors containing MyoD were constructed and their gene expression and ability to induce skeletal muscle gene expression within cardiac fibroblasts in vitro was determined by immunostaining using the muscle-specific marker alpha-actinin. The MyoD adenovirus vector was proposed to be more effective in vitro at converting fibroblasts to a muscle phenotype compared to the retroviral vector. This was confirmed by cardiac fibroblasts infected with recombinant MyoD adenovirus showing muscle-specific features such as sarcomeres when examined at the ultrastructural level by electron microscopy. The adenovirus vector was used to infect cells within an area of myocardial infarct in a rabbit model in vivo. Immunocytochemistry analysis confirmed that the adenovirus vector expressed MyoD protein and also the muscle-specific marker, skeletal myosin heavy chain. The injection of MyoD adenovirus can lead to the expression of skeletal muscle within an area of infarct. Co-injection of MyoD and beta-galactosidase adenoviruses enabled the localisation of expression of both exogenous genes within the infarct. This revealed that the area of skeletal muscle expression corresponded to the area of viral injection, therefore the muscle generation resulted from the expression of exogenous MyoD. There are also alterations in the signal transduction pathways within affected cardiac myocytes. Phosphodiesterase inhibitors have been used as positive inotropic agents in heart failure patients. Recombinant adenoviruses were constructed which expressed the type IV phosphodiesterases RDl and rPDE6 under the control of the CMV promoter. By overexpressing the phosphodiesterases RDl and rPDE6, the long term aim is to investigate whether contractility may be decreased and the inotropic state of the heart regulated in such a way that overall cellular energy levels within the myocardium could be conserved. The ultimate long term aim of this part of work would be reduce the level of type IV phosphodiesterases in the cell, using antisense constructs for rPDE6 sequences, and consequently augment the effects of the cAMP by preventing its breakdown and thereby sustain its level within the cell. The short term aim of this work was to produce functional adenoviral vectors which expressed the type IV phosphodiesterases, RDl and rPDE6. This was achieved and demonstrated by Western blotting and cAMP assays. This project has formed the basis for future work into the role of recombinant PDE expression in ischaemic tissue.
Notlar:
School code: 0547
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
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XX(684809.1) | 684809-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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