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Priprava enkapsuliranih celic za dostavo rastnih dejavnikov
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Kadunc, Lucija
(
Author
),
ID
Hafner Bratkovič, Iva
(
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)
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,
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Jerala, Roman
(
Comentor
)
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Abstract
Rastni dejavniki so pomembni regulatorni proteini, ki vplivajo na celično rast, migracijo in diferenciacijo ter posledično pospešijo procese celjenja ran. Rastne dejavnike sproščajo številne celice, med drugim tudi matične. V okviru doktorske naloge smo pripravili celično napravo, ki sprošča rastne dejavnike in druge zdravilne učinkovine, ki izboljšajo celjenje in regeneracijo tkiva. Za pripravo celične naprave smo stabilne celične linije, ki izločajo različne terapevtske molekule, enkapsulirali v votlo vlakno. Tako pripravljena celična naprava omogoča lokalno tvorbo zdravilnih učinkovin s strani enkapsuliranih celic, hkrati pa preprečuje stik med celicami in imunskim sistemom gostitelja. Analizirali smo sekretom mešanice stabilnih celičnih linij in potrdili, da celice izločajo proteine, ki pospešijo celjenje. Z uporabo celičnih modelov v pogojih in vitro smo potrdili biološko aktivnost rastnih dejavnikov, ki jih izločajo stabilne celične linije. S sekveniranjem mRNA tarčnih celic smo podrobnjeje ovrednotili njihov odziv na dodatek rastnih dejavnikov. Delovanje celične naprave smo testirali v pogojih in vivo ter pokazali, da je terapevtska naprava pospešila celjenje rane v primerjavi s kontrolo. Že po sedmih dneh je prišlo do povečanja tvorbe granulacijskega tkiva in obnovitve kožne bariere. Za boljše posnemanje naravnega procesa celjenja smo pripravili izboljšano inducibilno celično napravo. To nam omogoča, da z uporabo zunanjega signala preklopimo med dvema različnima kombinacijama sproščenih rastnih dejavnikov, ter posledično boljši nadzor nad izločenimi zdravilnimi učinkovinami. Pripravljena inducibilna celična naprava je v pogojih in vivo izboljšala hitrost celjenja rane v primerjavi s kontrolo, kar potrjuje njen pozitiven terapevtski vpliv ter potencial za uporabo v regenerativni medicini. Vendar pa uporaba viabilnih celic, ki jih vstavimo v telo, vedno predstavlja določeno tveganje. Za izboljšanje celične terapije bi lahko v celice vnesli dodatni varnostni mehanizem. Eden takšnih je vnos prezgodnjih terminacijskih kodonov v določene gene, tako spremenjeni geni pa se lahko izražajo le ob dodatku določenih sintetičnih spojin. Z uporabo poročevalskih proteinov smo testirali učinkovitost različnih sistemov, ki omogočajo preskok prezgodnjega terminacijskega kodona. Potrdili smo delovanje sistema aminoacil tRNA-sintetaze skupaj s supresorsko tRNA in sintetično aminokislino (Bpa). Pokazali smo, da pride do neželenega izražanja poročevalcev z enim kodonom TAG v odsotnosti sintetične aminokisline, zato smo pripravili poročevalec z dvema prostorsko ločenima kodonoma stop. S tako izboljšanim poročevalcem nismo zaznali puščanja sistema. Na podlagi tega smo sklepali, da bi lahko pristop z dodatkom kodona stop na 5' mestu uporabili kot strategijo za nadzor izražanja genov.
Language:
Slovenian
Keywords:
celična naprava
,
rastni dejavniki
,
celjenje ran
,
preskok prezgodnjih terminacijskih kodonov
,
varnostni mehanizem
Work type:
Doctoral dissertation
Organization:
MF - Faculty of Medicine
Year:
2020
PID:
20.500.12556/RUL-117271
COBISS.SI-ID:
22066179
Publication date in RUL:
04.07.2020
Views:
1414
Downloads:
289
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Language:
English
Title:
Development of encapsulated cells for growth factor delivery
Abstract:
Growth factors are important regulatory proteins that affect cell growth, migration and differentiation, consequently influencing healing processes. They are secreted by numerous cells including stem cells. Here we present a cell device that secretes growth factors and other therapeutic molecules for improvement of tissue regeneration. Cell device was prepared by encapsulating stable cell lines secreting therapeutic molecules into hollow fiber enabling in situ production of growth factors while protecting cells from host immune system. Secretome analysis of cell mixture revealed upregulation of proteins involved in wound healing. Cell response to addition of conditioned media was evaluated by RNA sequencing of target cells. In vitro testing confirmed biological effect of produced growth factors. Cell device was tested using in vivo skin wound healing mouse model. In the presence of therapeutic device healing significantly improved compared to control and skin wound barrier was restored in 7 days with newly formed granulation tissue. To better recapitulate healing process we designed improved inducible cell device that can switch between production of different therapeutic molecules giving us even better control over the set of secreted factors. In vivo inducible device significantly improved healing compared to control, confirming its therapeutic effect. Taken together, results show a potential of a new cell device for use in regenerative medicine. Significant safety considerations are present when introducing living cells into the body. To improve safety of cell therapy, a promising approach could be introduction of a premature stop codon (PTC) in a selected gene that allows its expression only in the presence of non-canonical amino acids. Different strategies of amber PTC read-through in mammalian cells were tested. The incorporation of a tRNA synthetase together with a TAG codon-specific tRNA achieved PTC read-through depending on the addition of a non-canonical amino acid (Bpa). While single TAG codon incorporation exhibited low but detectable expression of the reporter protein even in the absence of Bpa regardless of its position, introduction of a double PTCs, including additional 5’-PTC position enabled virtually leakage-free functional gene translation. Introduction of an additional 5’ PTC therefore represents a generally applicable strategy.
Keywords:
cell device
,
growth factors
,
wound healing
,
premature stop codon readthrough
,
safety mechanism
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