| ObjectiveNeural crest stem cells (NCSC) originated from dorsal neural tube in the embryonic period. Part of the NCSC directly differentiated into the neurons and glial cells of the enteric nervous system (ENS), known as the gut neural crest stem cells (GNCSC). The developmental abnormalities in the GNCSC will lead to the enteric nervous system diseases, and their number, function and different types of the cell play a key role during ENS development. Some studies confirm that in patients with anorectal malformations exist nervous system abnormalities, congenital megacolon, is also a congenital enteric nervous system abnormalities result. The discovery and success in vitro culture of GNCSC, can help us use GNCSC stem cell as transplantation, which can completely cured Hirschsprung's disease and solve the incontinence and other issues after operations in children with anorectal malformations, which might be a promising therapy.Although in recent years, articles on the GNCSC in vitro is not unusual, but some key steps in the GNCSC in vitro remained obscure, and there is no consensus in the uniform standard choice of materials. This experiment aimed at improving the method in GNCSC in vitro culture and comparing the difference in GNCSC growth disassociated from different gestational age rat embryos and postnatal rats, and exploring the best material choice. At the same time by immunofluorescence staining technology,we investigated the expression of Wnt5a in the proliferation and differentiation of GNCSCs to study the role of Wnt5a signling in the intestinal diseases caused by neurodevelopmental abnormalities.In recent years research has shown that the highly conserved Wnt signaling protein family in embryonic development play an important role in signaling through different molecular interactions, Wnt proteins trigger the regulation of cell growth, migration, differentiation and development, and many other complex signaling cascade reaction。In this family, research about Wnt5a become more and more, one of which reported that Wnt5a played an important role in proliferation and differentiation of GNCSC in the CNS. In this paper,we research the expression of Wnt5a in GNCSC proliferation and differentiation by immunofluorescence staining and whether the Wnt5a perform a key role in the formation and development of the ENS.Methods1. Isolation, culture, charactrization of GNCSC from different gestational age ratsIn order to perform GNCSC primary culture,we select microsurgical instruments to extract gut from rats 2 days post natal and the embryos,which were harvested via cesarean section on E15 to E21. We do the subculture after neurosphere-like structures (neurosphere-like bodies, NLB) appears in primary culture. And use GNCSC 5-7 days after subculture to do immunofluorescence identification. And compare the growth of NLB from different embryonic days.2. Wnt5a expression in the proliferating and differentiating GNCSCGNCSC isolated from rat embryo of E19 divided into 5 groups 6 days after subculture. Group 1 was directly used for immunofluorescence staining, the other groups were seeded on the coverslip in induced differentiation medium containing serum and then immunocytochemitry 1 day,3 days,5 days,7 days post inducing differentiation.Results1. By comparing the time of neurospheres'formation in different gestational age and after birth, we discovered that, the neurospheres appeared on the third day after primary culture which extracted from E17, E19.And it appeared on the fifth day after primary culture which extracted from E21, P2.2. There was a strong Wnt5a expression in GNCSC undifferentiated and in GNCSC the first day after differentiation.but on the third day,the expression decreased, and there is almost no expression observed on the days 5-7. Conclusions1. When we extract GNCSC from rat embryo,we should choose the E19 rat.2. Wnt5a plays an important role during the GNCSC proliferation and early differentiation.
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