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Browsing by Subject "verkkokalvon pigmenttiepiteeli (RPE)"

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  • Non-viral gene delivery : Carrier-mediated transfection to retinal pigment epithelial cells and endothelial cells 
    Viljamaa, Matleena (2015)
    Gene therapy involves the delivery of exogenous DNA into the target cells in order to produce therapeutic protein or to correct a genetic defect. The use of cationic liposomes and polymers as carriers of DNA is based on observations that positively charged carriers bind to anionic DNA protecting its premature degradation and facilitating its cellular uptake in transfection. The modification of carriers and the engineering of DNA are proposed to enable efficient and prolonged protein expression after transfection. Gene therapy is a potential treatment for age related macular degeneration (AMD). The dysfunction of retinal pigment epithelial (RPE) cells is assumed to be a significant factor in the development of AMD. The aim of this Master's thesis was to study non-viral gene delivery to RPE cells and endothelial cells using several carrier/DNA combinations. Carriers in this study were DOTAP/DOPE/PS liposomes, methacrylamide based (PDMAEMA) micelles, and anionic lipid coated DNA complexes (LCDCs). The carriers were complexed with episomal plasmid DNA or minicircles using secreted alkaline phosphatase (SEAP) gene as a marker gene. Adult retinal pigment epithelial (ARPE-19) cells, human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE), human embryonic primary RPE cells and endothelial cells (EaHy 926) were used in transfections. In ARPE-19 cells linear PBuA-PDMAEMA -based complexes reached the transfection efficiency of positive control whereas in human primary RPE cells star-like PBuAPDMAEMA -based complexes were the most efficient. In human primary RPE cells, SEAP secretion lasted at least 18 days when PDMAEMA-based micelles complexed with plasmid or minicircle with cytomegalovirus (CMV) promoter were used. High nitrogen/phosphate (n/p) ratios of polyplexes decreased cell viability. DOTAP/DOPE/PS/DNA lipoplexes transfected EaHy cells with high efficiency. In hESC-RPE, lipoplexes also exceeded the transfection efficiency of the positive control and the marker protein secretion lasted ~20 days. Human elongation factor 1a (EF1a) promoter could not prevent transgene silencing. Gene delivery did not succeed with LCDCs in any transfection. According to the results, PBuA-PDMAEMA-polymers and DOTAP/DOPE/PS-liposomes complexed with episomal plasmid or minicircles are potential gene delivery agents for further studies in AMD. More investigation is needed i.e. to confirm the transfection efficiency of the complexes in non-dividing cells.
  • Verkkokalvon pigmenttiepiteelin transportterien tutkiminen solumalleilla ja kanin silmäkudoksella (Kirjallisuusosa: OATP-transportterit sarveiskalvolla ja veri-silmäesteissä) 
    Paavilainen, Nea (2024)
    The eye is well-protected by several anatomical and physiological barriers which also pose significant challenges for ocular drug delivery. Even though ocular pharmacokinetics and the permeability of eye’s important anatomical barriers, such as the cornea and the blood-ocular barriers, have been thoroughly investigated, the significance of active transport in the eye is not completely understood. It is known that several drug transporters are also expressed in ocular tissues, but scientific information on this area is still dispersed and incomplete. The aim of the literature review in this master’s thesis was to compile the current knowledge on the expression and activity of OATP transporters (SLCO; organic anion transporting polypeptides) in cornea and in the blood-ocular barriers. Main principles of ocular pharmacokinetics and common methods for studying transporters are also discussed. The experimental part in this thesis is focused on retinal pigment epithelium (RPE) which is a substructure of the blood-retinal barrier. The transporters of the RPE were studied with three different RPE model systems: human RPE cell line (ARPE-19), fetal primary RPE cells (hfRPE; human fetal retinal pigment epithelium) and ocular tissues of the rabbit. In detail, transporter expression was studied with proteomics from the plasma membrane of isolated rabbit RPE and transporter activity by cellular uptake assays (ARPE-19, hfRPE) in vitro and permeability experiments with rabbit RPE-choroid-sclera ex vivo. As with the literature part, the experimental work was mainly focused on the human and rabbit OATP/Oatp transporters. In this thesis, ten important drug transporters were detected from the rabbit RPE. No significant OATP/Oatp activity was observed either in vitro or ex vivo experiments so these transporters seem not to have a great role in the disposition of their substrates in the studied RPE models. However, signs of other active transport were evident especially in the ARPE-19 cell line, in which significant accumulation of the tested substrate, 4’,5’-dibromofluorescein (DBF), was noted in the presence of several inhibitors. The phenomenon was suspected to result from efflux inhibition, but the responsible transporters could not be unequivocally detected. In conclusion, the findings of this thesis highlight the importance of conducting further research on the transporters of the RPE and choosing a suitable RPE model case-by-case for each study. With the compounds used in this thesis, ARPE-19 and hfRPE cells showed marked differences in efflux activity while the small size and fragile structure of the posterior ocular tissues of the rabbit caused notable difficulties in performing the transporter studies.

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