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Izražanje fragmentov protiteles proti prionom v mezenhimskih matičnih celicah in njihova karakterizacija
ID Novak, Neža (Author), ID Rajčević, Uroš (Mentor) More about this mentor... This link opens in a new window

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Abstract
Terapija prionskih bolezni še ni poznana, se pa v zadnjem času raziskuje veliko novih principov zdravljenja. Eden od teh je zdravljenje s protitelesi proti prionom, ki bi lahko preprečila širjenje PrPSc. Mezenhimske matične celice (MMC) so potencialni vektor za dostavo teh protiteles v možgane zaradi lastnosti samoobnavljanja, diferenciacije, imunomodulacije, sposobnosti prečkanja krvno-možganske pregrade ter pato-tropizma. V naši raziskavi smo skušali vstaviti zapis za fragment scFv protitelesa V5B2 v mišje MMC s pomočjo elektroporacije. V prvem delu smo optimizirali pogoje elektroporacije, in sicer smo uporabili 8 kvadratnih pulzov, dolgih 1 ms, z močjo električnega polja 1,0 kV/cm in frekvenco 1 Hz. Celice v koncentraciji 3 x 106 celic/ml smo porirali v visoko prevodnem pufru. Koncentracija plazmida z vstavljenim GFP je bila 30 µg/ml. Elektroporacijo smo ponovili s plazmidi z vključki za znotraj- in zunajcelično izražanje scFv V5B2. Celice smo okarakterizirali s pretočno citometrijo in ugotovili, da ima elektroporacija le majhen vpliv na fenotip MMC. Prisotnost scFv V5B2 v celicah in gojišču smo skušali dokazati z imunocitokemijsko analizo in testom ELISA, a nismo bili uspešni. Glede na naše rezultate se scFv V5B2 ne izraža v mišjih MMC. Za uspešno izražanje bi bila potrebna nadaljnja optimizacija metode transfekcije, morebitna zamenjava vektorja konstruktov ter optimizacija analitičnih metod za detekcijo izražanja.

Language:Slovenian
Keywords:prioni, mezenhimske matične celice, protitelo, elektroporacija
Work type:Master's thesis/paper (mb22)
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2017
Publisher:[N. Novak]
UDC:602.9:602.621:616-097.3(043.2)
COBISS.SI-ID:8744313 This link opens in a new window
Publication date in RUL:11.06.2017
Views:1255
Downloads:762
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Secondary language

Language:English
Title:Expression of anti-prion protein antibody fragments in mesenchymal stem cells and their characterization
Abstract:
There is no known therapy for treatment of prion diseases, but recently a lot of new principles of treatment have been discovered. One of these are anti-prion antibodies which could prevent spreading of PrPSc. Mesenchymal stem cells (MSC) are potentially a delivery system for these antibodies to the brain, due to their self-renewal, differentiation, immunomodulation properties, pathotropism and ability to cross the blood-brain barrier. In this thesis, we attempted to insert a molecular construct for scFv fragment of anti-prion antibody V5B2 into mouse MSC by electroporation. In the first part, we optimized electroporation parameters, by using 8 square wave pulses, 1 ms long, with 1,0 kv/cm electric field strength and 1 Hz frequency. Cells were resuspended in high conductivity electroporation buffer to a final concentration of 3 x 106 cells/ml and 30 µg/ml of GFP plasmid was added. Electroporation was repeated with plasmids containing intracellular and secretory scFv V5B2. The cells were characterized by flow cytometry where, after electroporation, only a small difference in phenotype of mouse MSC was observed. We tried to determine the expression of scFv V5B2 in and outside of cells by immunocytochemistry and ELISA, but were not successful. According to our results, the scFv V5B2 is not expressed in mouse MSC. Further optimization of transfection methods, possible replacement of vectors and optimization of analytical methods for detection of expression is required.

Keywords:prions, meschenchymal stem cells, antibody, electroporation

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