Expression of neural adhesion molecules (NCAM) in healthy and diseased renal tissue
Ekspresija neuralnih adhezionih molekula (NCAM) u zdravom i obolelom bubrežnom tkivu
Author
Ćirović, SanjaMentor
Marković-Lipkovski, JasminaCommittee members
Müller, Claudia A.Vasiljević, Jovan
Tatić, Svetislav
Metadata
Show full item recordAbstract
Introduction: Kidney performs essential physiological roles that include
metabolic waste excretion and maintenance of fluid and electrolyte balance. Different
factors may lead to renal dysfunction. Kidney diseases are currently a global public
health problem. Fortunately, dialysis and transplantation provide life-saving treatments,
but these therapies are rife with limitations. Thus, new ways of therapies, such as stem
cell treatment are more than required as an alternative and new glimmer of hope to all
nephrology patients. Neural cell adhesion molecule (NCAM) is widely expressed during
kidney development and it presents potential marker for renal stem/progenitor cells.
Aim: Aim of this dissertation was evaluation of NCAM presence in different
types of renal tissue (normal fetal or adult, and pathologically changed), and comparison
and correlation of NCAM expression with already defined stem cell markers in order to
more closely characterize renal fetal progenitor cells.
M...ethod: Specifically designed primers were used for reverse transcriptase PCR,
after mRNA isolation from fetal, neonatal, adult normal, adult tissues with interstitial
fibrosis and tumor tissues, to clarify presence of different NCAM on nucleotide level.
Also, Western Blot tests were performed in order to identify expression of different
NCAM protein isoforms in healthy and kidney with disease. Further, FACS analysis
was done on renal cell lines to correlate NCAM expression with adult renal stem cell
markers CD24 and CD133. Co-expression and co-localization of NCAM with other cell
surface markers was also examined using immunohistochemistry and double
immunofluorescent staining.
Results: Our results showed aberrant NCAM expression in renal tumor tissues
as well as in tissue with interstitial fibrosis. Interestingly in both types of mentioned
tissues NCAM had co-expression with FGFR1, tyrosine kinase receptor, responsible for
cell proliferation and aggressive behavior in some epithelial tumors. Considering
NCAM expression on fetal samples possible renal progenitor population cell pool of
NCAM+CD24+ cells was found, which was not the case with NCAM+CD133+ cell pool.
Also, we had detected that NCAM molecules were post-trasnaltionally modified...
UVOD: Bubreg ima značajnu fiziološku ulogu u izlučivanju štetnih produkata
metabolizma putem urina, kao i ulogu u održanju ravnoteže vode i elektorlita. Različiti
faktori mogu dovesti do poremećaja funkcije bubrega. Bolesti bubrega su trenutno
globalni problem javnog zdravlja. Dijaliza i transplantacija su trenutno dominantni
vidovi terapije koje se primenjuju u lečenju bubrežne isuficijencije, ali uz puno
ograničenja. Novi načini terapije, kao što je tretman matičnim ćelijama, su više nego
potrebni kao alternativa standardnim metodama lečenja i predstavljaju novi tračak nade
za sve pacijente koji imaju neki oblik bubrežne disfunkcije. Neuralni ćelijski adhezioni
molekul (NCAM) je široko ispoljen tokom razvoja bubrega i predstavlja potencijalni
marker renalnih stem/progenitorskih ćelija.
CILJ: Cilj ove disertacije je evaluacija NCAM ekspresije u različitim tipovima
bubrežnog tkiva (fetalno, adultno normalno i adultno tkivo sa intersticijskom fibrozom i
tumorsko tkivo), kao ...i njena korelacija sa ekspresijom već definisanih markera renalnih
stem ćelija kako bi se preciznije definisale fetalne renalne progenitorske ćelije.
METOD: Detekcija različitih NCAM izoformi na nivou nukleotida je vršena
primenom RT-PCR metode, nakon izolacije iRNK iz fetalnog i normalnog adultnog
tkiva, kao i tumorskog i tkiva sa intersticijskom fibrozom. Prisustvo NCAM isoformi na
proteinskom nivou, u pomenutim tkivima detektovano je upotrebom Western blot-a i
Imunoprecipitacije. Ko-ekspresija NCAM-a sa CD24 i CD133, markerima adultnih
renalnih progenitora ispitivana je upotrebom protočne citomertije (FACS) na renalnim
ćelijskim linijama. Imunohistohemijskim i dvostrukim imunofluorescentnim bojenjem
ispitivana je koekspresija i kolokalizaciji NCAM-a sa drugim ćelijskim markerima.
REZULTATI: RT-PCR rezultati ove doktorske disertacije su pokazale da sve
ćelijske linije i ispitivana tkiva, osim tumorskog tkiva eksprimiraju tri glavne NCAM
isoforme NCAM-120, NCAM-140 i NCAM-180. Takođe, detektovana je aberentnaprekomerna
NCAM ekspresija u tumorskom, kao i u tkivu sa intersticijskom fibrozom.
Interesantan nalaz čini pokazana koekspresija NCAM-a sa FGFR1, tirozin kinaznim
receptorom, koji ima ulogu u ćelijskoj proliferaciji i agresivnom ponašanju pojedinih
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epitelnih tumora.
Faculty:
Универзитет у Београду, Медицински факултетDate:
13-03-2015Projects:
- Characterisation of human renal stem/progenitor cells: identification of new cell surface markers of renal multipotent cells which could have regenerative role in kidney lesions (RS-MESTD-Basic Research (BR or ON)-175047)