| BackgroundNeural tube defects(NTDs)are a serious congenital defect of the nervous system.Due to an abnormal neural tube closure during the embryonic period,they mainly manifestallkinds of brain malformations,and recessiveness.Spina bifida and so on.Neural epithelial cells,neural stem cells(NSCs),are the key cells of neural tube closure and cover the surface of neural tube.The proliferation,differentiation,and migration of neural stem cells(NSCs)are the key to the normal closure of neural tubes.Their abnormal proliferation and differentiation can cause neural tube.Defects,which have the ability to self-renew and differentiate into multiple cells,can differentiate into various nerve cells.In various regulatory pathways,Notch signaling pathway plays a sophisticated and complex role in cell proliferation and differentiation and embryonic development.Notchl is an important molecule in the Notch family of proteins and plays a key role in the development of the central nervous system.Notchl Signaling pathways are activated by the action of gamma endocrine enzymes.All-trans Retinoic Acid(ATRA)is the normal metabolite of retinoic acid in the body and is an important factor in embryonic development.Studies have found that excessive all-trans retinoic acid lead to NTDs,but its specific molecular mechanism is not clear.In this study,the role of Notch 1 in all trans-retinoic acid-treated NSCs was studied from the cytological level to investigate the regulatory effect of Notchl on the proliferation and differentiation of NSCs,and to reveal the molecular mechanism of all-trans retinoic acid-induced neural tube defects.This provides a new direction for the application of neural stem cells and clinical prevention of neural tube defects.ObjectiveBy using ATRA-treated neural stem cellsto detect the neural stem cell differentiation of cell markers and Notchl expression,lnvestigate the role of Notchl and ATRA in differentiation of neural stem cells and investigate the role of Notchl in all-trans retinoic acid-induced differentiation of neural stem cells,thus revealing its effect on neural tube development.Methods1.Primary neural stem cell extractionC57/BL6 mice were divided into male and female rats at a ratio of 2:1(2 females and 1 male).The female rats were checked for thrombus at 8 o'clock inthe morning on the second day.The thrombus was recorded as 0.5 days of pregnancy.The primary neural stem cells were extracted from the brains of pregnant 18.5 fetuses.2.ImmunofluorescenceNeural stem cells were induced to differentiate with a differentiation medium containing 10%fetal bovine serum,and then neural stem cells and differentiatedneurons,astrocytes,and oligodendrocytes were identified by immunofluorescene,and to detect the distribution of Notch 1 in these cells.3.Western blotting3.1 Notchl with Nestin,NF,GFAP and GALC(They are markers of neural s-tem cell,neurons,astrocytes and oligodendrocytes respectively)were identified by Western blotting in 1,3,5,7 days respectively.thatthe cells were cultured in normal differentiation media and the media with 1 ?mol/L ATRA.3.2The neural stem cells were divided into four groups:normal differentiation media group,normal differentiation media + DMSO group,normal differentia-tion media+DMSO+25?Mol y-secretase inhibitor group,nonnal differentiation media+DMSO+50?Mol y-secretase inhibitors group,then the expression of Notchl in each group was detected by Western Blotting.Results1.When the primary neural stem cells were cultured for one day,multiple neurosphere formations were seen.At 3 days of differentiation,the morphology of the neurospheres began to change and some of the neural stem cells were released from the neurospheres.On the 5th day of differentiation,the neurospheres were significantly reduced,protruding out of the cords.On the 7th day of differentiation,there are many different forms of nerve cells.One is a round or oval cell body with a long-swelled neuron-like cell,and the other is a cell body with a polygonal shape and a number of thicker stretches.The protrusions showed star-like glial-like cells with obvious nuclei,and one was oligodendrocyte-like cells with small cell bodies and protruding short protrusions.2.Nestin,NF,GFAP,and GALC were positive in neural stem cells in differentiation medium by immunofluorescence,and Notch1 was present in the neural stem cells,neurons,astrocytes and oligodendrocytes3.The contents of Notch1,NF,GFAP,and GALC before and after neural stem cell treatment were changed from 1 day to 5 days.Notch1 was found in the neural stem cells of the control group,and there was a significant increase from the first day to the fifth day(P<0.05).From the fifth day to the seventh day,Notch1 was significantly decreased(P<0.05),NF was significantly increased on the third day compared with the first day(P<0.05),and decreased on the fifth and third days(P<0.05).The contents of GFAP and GALC did not change significantly on days 1 and 3,and GFAP on day 5 was significantly lower than on day 1(P<0.05),while the content on day 7 was significantly higher than that on day 5(P<0.05).GALC on the 5th day was significantly lower than on the 1st day,and the content on the 7th day was significantly increased compared with the 5th day(P<0.05).In the experimental group,there was no significant change in Notch1 from day 1 to day 3 in the neural stem cells of the experimental group.The content of Notch1 was significantly decreased on the 7th day compared with the 1st day(P<0.05),while the content of NF on the 7th day was significantly increased compared with the 1st day(P).<0.05),Compared with the first day,the 5th day was significantly increased(P<0.05).The content of GFAP on the 7th day was significantly increased compared with the 1st day(P<0.05).The 5th day was significantly increased compared with the 1st day.(P<0.05),the content of GALC on the seventh day was significantly increased compared with the first day(P<0.05),and the fifth day was significantly increased compared with the first day(P<0.05).4.Changes of Notchl content in neural stem cells treated with y-endocrine inhibitors The expression of Notchl in the intracellular domain(molecular weight 120KD)in group C was significantly lower than that in group A(P<0.05).The neural stem cells Notchl in group D were intracellular The expression level of segmental(molecular weight 120KD)was significantly lower than that of group A(P<0.05).The expression level of Notchl in the C group was significantly higher than that in group A(P<0.05).The neural stem cell Notchl in group D was significantly decreased.The intracellular segment(molecular weight 300KD)expression was significantly more than that in the A group(P<0.05).Conclusions1.Notchl inhibits neural stem cell differentiation.2.After ATRA treatment of neural stem cells,the content of Notchl decreased and the differentiation of neural stem cells increased.ATRA promoted the differentiation of neural stem cells.3.Neural stem cell treated with y-Secretase inhibitors,that reduce the content ofNICD(Notch intracellular domain,NICD),decrease the ability that to inhibit the differentiation of neural stem cells.We speculate that ATRA mainly inhibits theNotchl signaling pathway by inhibiting the y-Secretase,thereby promoting the differentiation of neural stem cells.It provides us with a the-oretical basis for the future clinical application of neural stem cells and provides us with a new treatment window for the prevention of fetal neuraltube defects. |
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