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Uporabnost nano-protiteles za razvoj ciljanega zdravljenja glioblastoma
Šamec, Neja (Author), Komel, Radovan (Mentor) More about this mentor... This link opens in a new window

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Abstract
Glioblastom multiforme (GBM) je gliom, tumor osrednjega živčnega sistema, najpogostejša oblika možganskega tumorja, za katerim zboli 3-5 bolnikov na 100.000 ljudi. Povprečni čas preživetja bolnikov z GBM je od časa diagnosticiranja 12 do 18 mesecev, ob kombinirani postoperativni terapiji z uporabo temozolomida. Genetska heterogenost GBM je posledica glioblastomskih matičnih celic (GMC). GMC zaradi svoje odpornosti proti kemoterapevtikom in radioterapiji uspešno napadajo zdravo okoljsko tkivo. Zdravljenje je dodatno oteženo zaradi krvno-možganske pregrade (KMP), saj je prehod kemoterapevtikov skozi njo zelo otežen. Za izboljšanje zdravljenja GBM in izid za bolnika je potrebna stalna dostava zdravil v celice glioma, obenem pa je potrebno zmanjšati učinek zdravil na sosednje, zdrave nevrone in celice glije. Novi pristopi zdravljenja GBM so zato zelo zaželeni in potrebni. Za razvoj usmerjenega zdravljenja pa še vedno potrebujemo odkritje bolj specifičnih biomolekularnih označevalcev GMC, kot tudi njihovih tarčnih zdravil, ki bi prehajala KMP. To lahko dosežemo s proteomskim pristopom, ki temelji na nano-protitelesih, ki so eno-domenski antigen-vezavni fragmenti, pridobljeni iz kemelidnih težkoverižnih protiteles, ki zaradi svoje majhnosti lahko prehajajo KMP. V doktorski nalogi smo izdelali knjižnico nano-protiteles in opravili njihovo imunoafinitetno obogatitev, izvedeno na celotnih GMC, in tako pridobili nano-protitelo, specifično za bio-označevalec GMC. Z masno spektrometrijo smo ugotovili, da je nov bio-označevalec mitohondrijski translacijsko-elongacijski dejavnik TUFM. Diferenčno izražanje TUFM smo proučevali na ravni proteinov in mRNA, in sicer v celičnih linijah GBM (U87MG in U251MG), GMC in tkivih GBM, v primerjavi z izražanjem v nevralnih matičnih celicah (NSC) in normalnih možganskih tkivih. S prenosom western smo na ravni proteinov ter qPCR na ravni mRNA potrdili nadizražanje TUFM v GMC. Z imunohistokemijo, na tkivnih rezinah, vklopljenih v parafin, smo potrdili nadizražanje TUFM v tkivih GBM, v normalnem možganskem tkivu pa se TUFM ni izražal. Z imunocitokemijo smo potrdili vstop nano-protitelesa anti-TUFM v GBM celice U87MG, U251MG in GMC ter njegovo vezavo v območje mitohondrijev. Citotoksičnost vezave nano-protitelesa anti-TUFM na antigen smo preverili z metabolnimi testi na celičnih linijah GBM (U87MG in U251MG), GMC ter kontrolnih celičnih linijah – astrocitih, nevralnih matičnih celicah in človeških nesmrtnih keratinocitih. Nano-protitelo anti-TUFM je imelo citotoksičen učinek na vse celične linije GBM, medtem ko toksičnosti anti-TUFM na kontrolnih celičnih linijah nismo opazili. Z vklapljanjem nano-protiteles anti-TUFM v arheosome smo preverili ali obstaja boljši oz. bolj učinkovit dostavni sistem za vnos nano-protitelesa anti-TUFM v celice. Arheosomi niso citotoksični in vivo in imajo edinstvene strukturne lastnosti, ki so osnova za razvoj novih dostavnih sistemov zdravil. Te lastnosti so stabilnost pri visokih temperaturah, nizkem oz. visokem pH, odpornost proti fosolipazam in solem žolčnih kislin ter manjša membranska prepustnost. Nano-protitelesa anti-TUFM smo uspešno vklopili v arheosome in ugotovili, da le-ti vstopajo v celice GBM, U251MG in U87MG. Ugotovili smo, da ima nano-protitelo anti-TUFM, vklopljeno v arheosom, na celice GBM manjši citotoksični učinek kot ga ima samo nano-protitelo anti-TUFM. V doktorski nalogi smo dokazali specifičnost in izrazit zaviralni učinek nano-protitelesa anti-TUFM na rast GMC, kar bi lahko v prihodnje pripomoglo k razvoju specifičnega pristopa za zdravljenje glioblastoma.

Language:Slovenian
Keywords:glioblastom multiforme, bio-označevalec TUFM, nano-protitelesa, citotoksičnost
Work type:Doctoral dissertation (mb31)
Organization:MF - Faculty of Medicine
Year:2018
Views:635
Downloads:252
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Secondary language

Language:English
Title:Implementation of nanobodies for the design of glioblastoma targeting therapy
Abstract:
Glioblastoma multiforme (GBM) is a glioma, a tumor found in central nervous system. It is the most common form of brain tumors, which affects 3-5 patients per 100,000 people. The average survival period of patients with GBM is 12 to 18 months, which includes resection and combination of postoperative therapy with temozolomide. Glioblastoma stem cells (GMC) are responsible for high genetic heterogeneity of GBM, and due to their resistance to chemotherapy and radiotherapy they successfully invade healthy tissue. The treatment is further aggravated, due to difficult transition of chemotherapeutics through blood-brain barrier (BBB). To improve GBM treatment and the outcome of patients, it is necessary to continuously deliver drugs to the glioma cells while reducing the effect of drugs on adjacent, healthy neurons and glial cells. New GBM treatment approaches are therefore much needed. For the development of targeted GBM treatment, we still need the discovery of more specific GMC biomarkers and the corresponding targeting drugs that would pass BBB. This can be achieved by a proteomic approach based on nanobodies, single-domain antigen-binding fragments, derived from camelid heavy chain antibodies that can, due to their small size, pass BBB. In the doctoral thesis, we constructed a nanobody library and biopannings were made on the whole GMCs. We obtained a nanobody specific for a GMC antigen. Mass spectrometry determined that the new biomarker of GMC is the mitochondrial translational-elongation factor TUFM. Differential expression of TUFM was studied at the protein and mRNA levels in the GBM cell lines (U87MG, and U251MG), GMC and GBM tissue, compared to its expression in neural stem cells (NSC) and normal brain tissue. Western blot and qPCR confirmed the TUFM overexpression in GMC. With immunohistochemistry, on paraffin-embedded GBM tissue, we confirmed the TUFM overexpression, whereas the normal brain tissue was negative for TUFM. Immunocytochemistry confirmed the entry of anti-TUFM nanobodies to the U87MG, U251MG and GMC cells and its binding to mitochondria. The cytotoxic effect of anti-TUFM nanobodies on GBM-related cell lines (U87MG, U251MG and GMC), and on control cell lines (astrocytes, NSC and human immortal keratinocytes) was measured through metabolic assays. Anti-TUFM nanobody had cytotoxic effect on all GBM cell lines, while on the other hand no toxicity of anti-TUFM on control cell lines was observed. Anti-TUFM nanobodies were encapsulated to archeosomes and used to verify their delivery in cells. Aerheosomes are not cytotoxic in vivo and have unique structural properties for the development of new drug delivery systems. These properties are stability at high temperatures, extreme pH values, resistance to phospholipases and bile salts and lower membrane permeability. Successfully, anti-TUFM nanobodies were encapsulated in the archeosomes and were found that they could enter the U251MG cells. Therefore, an encapsulated anti-TUFM nanobody exhibited lower cytotoxic effect on GBM cells than the anti-TUFM nanobody itself. In the doctoral thesis, we showed the specificity and pronounced inhibitory effect of anti-TUFM nanobody on GMC growth, which could in the future contribute to the development of a specific approach for the treatment of glioblastoma.

Keywords:Glioblastoma multiforme, Biomarker TUFM, Nanobodies, Cytotoxicity

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