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Wyświetlanie 1-10 z 18
Tytuł:
Optimization of a retroviral vector for transduction of human CD34 positive cells
Autorzy:
Szyda, Anna
Paprocka, Maria
Krawczenko, Agnieszka
Lenart, Katarzyna
Heimrath, Jerzy
Grabarczyk, Piotr
Mackiewicz, Andrzej
Duś, Danuta
Tematy:
transduction optimization
retroviral vector
CD34+ cells
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1041181.pdf  Link otwiera się w nowym oknie
Opis:
Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37°C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Latencja CMV - trudności w jej wykrywaniu u dawców przeszczepu komórek hematopoetycznych.
CMV latency - difficulties in detection in hematopoietic stem cells donors
Autorzy:
Wróbel, Justyna
Opis:
Ludzki wirus cytomegalii (HCMV) występuje powszechnie w populacji, nie wywołując zazwyczaj poważnych objawów. Posiada on zdolność do przechodzenia w stan latencji (uśpienia). Jest wtedy obecny w obrębie komórek gospodarza, ale nie zachodzi produkcja wirionów wirusowych. Głównym miejscem latencji są hematopoetyczne komórki progenitorowe CD34+. Wraz z tymi komórkami może dostać się do organizmu biorcy szpiku kostnego, gdzie może dojść do reaktywacji wirusa i w konsekwencji doprowadzić do wystąpienia szeregu groźnych objawów. Możliwa jest również sytuacja, w której wirus, obecny już wcześniej w komórkach biorcy, po transplantacji ulega reaktywacji pod wpływem immunosupresji, jakiej poddawani są biorcy przeszczepów. Dlatego ważne jest opracowanie skutecznej metody wykrywania latentnej formy HCMV. W niniejszej pracy podjęto próbę detekcji jego DNA metodą nested PCR oraz PCR w czasie rzeczywistym, jak również detekcji specyficznych dla latencji mRNA. Napotkano jednak przy tym sporo trudności.
Human cytomegalovirus (HCMV) is common in the population, usually without causing severe symptoms. It has the ability to establish latency. Virus is then present within the host cells, but without production of virions. The main sites of latency are hematopoietic progenitor cells - CD34+. Along with these cells HCMV may enter the organism of a bone marrow recipient, where it can reactivate virus and consequently lead to a number of serious symptoms. It is also possible the situation where a virus, already present in the cells in the recipient, is reactivated after transplantation under the influence of immunosuppression, which transplant recipients undergo. So it is important to develop effective methods of detection HCMV latency. In this study I was trying to detect latent HCMV genome by nested PCR and Real Time PCR, as well as detection of latency-specific mRNA. But I encountered many difficulties.
Dostawca treści:
Repozytorium Uniwersytetu Jagiellońskiego
Inne
Tytuł:
Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood
Autorzy:
Ołdak, Tomasz
Kruszewski, Marcin
Machaj, Eugeniusz
Gajkowska, Agnieszka
Pojda, Zygmunt
Tematy:
CD34+ cells
umbilical cord blood
green fluorescent protein
transfection
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1043723.pdf  Link otwiera się w nowym oknie
Opis:
Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34+ hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34+ hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34+ cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 μF, 550 V/cm, and 10 μg of DNA per 500 μl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34+ hematopoietic cells derived from human umbilical cord blood.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Effects of inhibitor of Src kinases, SU6656, on differentiation of megakaryocytic progenitors and activity of α1,6-fucosyltransferase
Autorzy:
Kaminska, Joanna
Klimczak-Jajor, Edyta
Skierski, Janusz
Bany-Laszewicz, Urszula
Tematy:
FUT8
Meg-01cell line
CD34+ cells
megakaryocytes
SU6656
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1040704.pdf  Link otwiera się w nowym oknie
Opis:
α1,6-fucosyltransferase (FUT8) attaches fucose residues via an α1,6 linkage to the innermost N-acetylglucosamine residue of N-linked glycans. Glycans with this type of structure are present in GpIIb/GpIIIa complex (CD41a) which is present on megakaryocytes (Mks) and platelets. CD41a is the earliest marker of megakaryocytopoiesis. The aim of this study was to analyse the morphology, phenotype, ploidy level and activity of FUT8 during induced differentiation/maturation of Mk progenitor cells in ex vivo culture. We used SU6656, a selective inhibitor of Src tyrosine kinases, as differentiation-inducing agent for Mks. The addition of SU6656 to the culture system of megakaryocytic progenitors from cord blood CD34+ cells and Meg-01 cell line induced their maturation towards later stages of Mk differentiation with increased activity of FUT8. We suggest FUT8 as a candidate for an early marker of differentiation and possibly of the ploidy level of Mks. We confirm a special status of FUT8 in megakaryocytopoiesis.
Dostawca treści:
Biblioteka Nauki
Artykuł
Tytuł:
Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy
Autorzy:
Markowicz, Sergiusz
Niedzielska, Joanna
Kruszewski, Marcin
Ołdak, Tomasz
Gajkowska, Agnieszka
Machaj, Eugeniusz
Skurzak, Henryk
Pojda, Zygmunt
Tematy:
CD34+ cells
umbilical cord blood
green fluorescent protein
electroporation
gene transfer
dendritic cells
Pokaż więcej
Wydawca:
Polskie Towarzystwo Biochemiczne
Powiązania:
https://bibliotekanauki.pl/articles/1041291.pdf  Link otwiera się w nowym oknie
Opis:
Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR+ cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34+ cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor α (TNF-α). Transfection of CD34+ cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.
Dostawca treści:
Biblioteka Nauki
Artykuł
Wyświetlanie 1-10 z 18

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