Abstract | Cilj: Utvrditi postoje li tijekom razvoja u spinalnim ganglijima čovjeka nediferencirane
stanice neuralnog grebena, analizirati prostorni i vremenski raspored neurona i njihovih
podtipova, pojavu potpornih stanica te utvrditi njihovu sposobnost umnažanja.
Metode i materijal: Imunohistokemijski i imunofluorescencijski analizirali smo tkiva 10
ljudskih plodova gestacijske dobi od 5. do 10. tjedana na parafinskim rezovima debljine 7µm.
Primarna protutijela na nestin, phox2b, PGP9.5, S100, GFAP, IB4 i NF200 prikazali smo
pomoću DAB-a i fluorescentnih sekundarnih protutijela (Texas Red, Fluorescein
izotiocijanat). U statističkoj analizi koristili smo t-test i Mann-Whitney test.
Rezultati: Svi istraživani čimbenici prisutni su u spinalnim ganglijima tijekom 5. do 10.
tjedna razvoja. Nestin i phox2b biljezi imali su najjači izražaj u 5. i 6. razvojnom tjednu (62%,
odnosno 73% pozitivnih stanica), postupno se smanjivali u 7. i 8., te u 9. i 10. (56%, odnosno
59% pozitivnih stanica). Dorzalni dio spinalnog ganglija imao je značajno veći postotak
nestina i phox2b u 7. i 8. tjednu gestacije (Mann-Whitney, p=0,012; t-test, p=0,007). Između
5. i 10. razvojnog tjedna, broj PGP9.5- (biljeg za neurone) pozitivnih stanica se postupno
povećavao s 41% na 55%, te je stalno bio veći u ventralnom dijelu ganglija. Broj NF200-
(biljeg mehanoreceptora) i IB4- (biljeg nociceptora) pozitivnih stanica bio je najniži u 5. i 6.
tjednu (9% odnosno 7% pozitivnih stanica). Tijekom 7. i 8. tjedna, broj NF200-pozitivnih
stanica se udvostručio (18%), a broj IB4-pozitivnih stanica se utrostručio (20%). Njihov broj
se tijekom 9. i 10. tjedna razvoja gotovo izjednačio (22% odnosno 24%). Tijekom cijelog
istraživanog razdoblja broj nociceptora i mehanoreceptora je bio veći u ventralnom dijelu
ganglija. Biljeg potpornih stanica GFAP bio je dvostruko veći (21%) od S100 biljega (11%) u
5. i 6. tjednu, a njihov se broj izjednačio u 7. i 8. tjednu (23% odnosno 28%). U 9. i 10.
tjednu, broj GFAP-pozitivnih stanica neznatno se povećao (25%), a broj S100-pozitivnih
stanica se učetverostručio u odnosu na 5. i 6. tjedan (40%). Između 5. do 10. tjedna postotak GFAP- i S100-pozitivnih stanica bio je veći u ventralnim nego u dorzalnim dijelovima
ganglija. Tijekom cijelog istraživanog razdoblja i nestin- i S100-pozitivne stanice
kolokaliziraju s biljegom proliferacije Ki-67. Neuroni (PGP9.5-pozitivne stanice) ne pokazuju
kolokalizaciju ni s Ki-67 ni s nestinom. Za GFAP i S100 postoji kolokalizacija u većini
ganglijskih stanica, dok za GFAP i nestin, te S100 i nestin, postoji kolokalizacija samo u
nekim stanicama.
Zaključci: Tijekom čitavog istraživanog razdoblja biljezi neuralnog grebena, neurona i
potpornih stanica pojavljuju se istodobno u spinalnom gangliju. Nezrele stanice se mitotski
dijele i služe kao zaliha multipotentnih stanica koje se mogu diferencirati u sve podtipove
stanica. Dok se neuroni ne dijele, potporne stanice se dijele, a njihov broj značajno raste
tijekom istraživanog razdoblja. Rano prisustvo nociceptora i mehanoreceptora, ukazuje na
mogućnost osjeta bola i dodira. Točan vremenski i prostorni slijed pojave pojedinih biljega u
stanicama, omogućuje diferencijaciju stanica spinalnih ganglija, dok promjene tog slijeda
mogu dovesti do patoloških poremećaja u oblikovanju i funkciji spinalnih ganglija. |
Abstract (english) | Aims: To determine the existence of undifferentiated neural crest cells in the spinal ganglia
during early human development, and analyse spatial and temporal distribution of neurons
and their subtypes, appearance of glial cells and their potential to divide.
Methods: Immunohistochemical and immunofluorescence techniques were performed on
tissues of 10 human conceptuses between 5th and 10th gestational weeks, using paraffin
sections. Primary antibodies to phox2b, PGP9.5, S100, GFAP, IB4 and NF200 proteins were
visualised using DAB or fluorescent secondary antibodies (Texas Red and Fluoresceinisothiocianate). T-test and Mann-Whitney test were used for statistical analysis.
Results: All investigated proteins were present in the spinal ganglia between the 5th and 10th
week of gestation. Nestin and phox2b were strongly expressed in the 5th and 6th
developmental week (62% and 73%, respectively), slightly decreasing during the 7th and 8th
week, and during the 9th and 10th week (56% and 59%, respectively). Dorsal parts of the
spinal ganglia in comparison to ventral parts had a significantly higher rate of nestin- and
phox2b-positive cells in the 7th and 8th week of gestation (Mann-Whitney, p=0,012 and
p=0,007 respectively). Between the 5th and 10th week of development, the number of PGP9.5-
positive cells was subsequently increasing from 41% to 55%, and was constantly higher in the
ventral parts of ganglia. The number of NF200- and IB4-positive cells was lowest in the 5th
and 6th developmental week (9% and 7% respectively). During the 7th and 8th week, number
of NF200-positive cells doubled (18%), while the number of IB4-positive cells tripled (20%).
Their number reached almost the same values (22% and 24%, respectively) during 9th and 10th
week. During the whole investigated period, the number of NF200- and IB4-positive cells was higher in the ventral parts of ganglia. GFAP marker for supporting cells was two fold higher
(21%) than S100 marker (11%) in 5th and 6th week. Their number nearly levelled in 7th and 8th
week (23% and 28% respectively). In the 9th and 10th week, the level of GFAP reached 25%,
while there were 40% of S100-positive cells. Between 5th and 10th week, the number of
GFAP- and S100-positive cells was higher in ventral parts of ganglia. Colocalization of nestin
and S100 with Ki-67 factor was observed during 5th to 10th week period, while PGP9.5 did
not colocalize with Ki-67 or nestin. GFAP and S100 colocalized in majority of ganglion cells,
while GFAP and nestin, as well as S100 and nestin, colocalized only in some cells.
Conclusions: During the whole investigated period, markers for neural crest cells and neural
and glial cells appeared simultaneously in the spinal ganglia. Undifferentiated cells
proliferated and served as a pool of multipotent cells that could differentiate into different cell
subtypes. While neurons did not divide, glial cells proliferated and their number significantly
increased during the investigated period. Early existence of nociceptors and mechanoreceptors
indicated possible appearance of pain and touch sensation. The expression of investigated
proteins changed in a temporally and spatially restricted pattern, thus enabling differentiation
of cell lines in the spinal ganglia. Changes in that pattern may lead to pathologic disturbances
of spinal ganglia formation and function. |