The Preliminary Research On The Role Of Ethionine In Neural Tube Defects

Objective:1.To detect whether ethionine intraperitoneal injection of C57BL/6 pregnant mice can cause fetal neural tube deformity.2.To isolate,culture and identify primary neural stem cells?NSCs?extracted from C57 mice,a cell model was provided for the study of ethionine-induced neural tube defects.3.By studying the effect of ethionine on the function of neural stem cells,the role of ethionine in the process of embryonic neural tube defects caused by C57BL/6 mice was studied.Methods:1.By way of intraperitoneal injection,ethionine was injected at a dose of 100 mg/kg into the mother day of C57BL/6 pregnancy 7.5 days?E7.5?,and then continued to feed to E9.5,E10.5 and E11.5.The intact embryos were extracted and separated,and the forebrain and hindbrain closure of E10.5 and E11.5 were observed by stereomicroscope and HE staining.The results of modeling were verified.2.Primary NSCs were extracted by mechanical method from C57 fetal rat brain tissue at E13.5 days of pregnancy,and then cultured in vitro by suspension cell culture.Immunofluorescence staining was used to first identify NSCs by detecting Nestin characteristic proteins of NSCs.Then,NSCs were induced to differentiate with DMEM/F12culture medium containing 10%FBS,and the characteristic proteins NF-H,MAP2,GFAP and GALC proteins of neurons,astrocytes and oligodendrocytes were detected by immunofluorescence staining.Perform NSCs differentiation and identification.3.The ethionine was added to the culture medium of the neural stem cells,and the experiment was divided into two groups:the control group?normally cultured NSC group?and the experimental group?with ethionine cultured NSC group?,and the two groups of cells were cultured.For a period of time,the cell morphology was observed under a fluorescence microscope and photographed.Under the image image analysis software,5-10 fields of each group were randomly selected for neurosphere counting and analysis,and the colony forming ability and nerve were observed.The formation ratio of the spheres and the growth state of the cells preliminarily infer the effect of ethionine on the proliferation of neural stem cells.4.Cell proliferation was measured in the experimental and control groups by the Cell Counting Kit-8 method?CCK-8 method?.5.The changes of cell apoptosis in the experimental group and the control group were detected by flow cytometry.6.The expression of Caspase 3 protein was detected by Western blotting to understand the effect of ethionine on the apoptosis of NSCs.7.The cell cycle changes of the experimental group and the control group were detected by flow cytometry.8.The expression of NF-H,MAP2,GFAP and GALC in the neurons,astrocytes and oligodendrocytes were detected by immunofluorescence in the experimental and control groups.9.Statistical analysis:Data were expressed as mean±standard deviation?X±S?,data were analyzed using SPSS 17.0 software,P<0.05 indicates statistically significant differences;data mapping was performed using GraphPad Prism 5 software and ImageJ-gray analysis software.Results:1.Ethyl ethionine was intraperitoneally injected into pregnant E57 C57BL/6 pregnant mice,and NTDs were successfully induced.The optimal dose was 100 mg/kg,and the incidence of NTDs was 54.7%.The NTDs in mice mainly showed craniofacial malformation and Stunting,other malformations include posterior forebrain neural tube,forebrain hypoplasia,developmental malformation,etc;HE staining showed that the cerebral vesicles were not closed and the structure was abnormal before and after fetal tuberculosis.2.The primary extracted NSCs grew well.Immunochromatographic staining of NSCs Nestin protein showed positive color development;after 7 days of induction of differentiation,neuron,astrocytes and oligodendrocyte characteristic proteins NF-H,MAP2,GFAP and GALC immunofluorescence staining Identification,also positive coloration.3.The experimental group and the control group had different colony forming ability and the ratio of neurosphere formation.The optimal concentration was 10mmol/L.The colony formation ability of the control group was significantly higher than that of the experimental group.The difference was statistically significant?12.60±1.074 vs.5.50±0.893,t=10.80,P<0.05?.Although the number of neurospheres with a diameter less than100um in the experimental group was higher than that in the control group,the number of neurospheres larger than 100um in diameter was much higher in the control group than in the experimental group?P<0.05?.4.The results of CCK-8 method showed that the absorbance value?OD?of the experimental group at 0h was not significantly different from that of the control group,and the next 1-5 days.The OD value was significantly lower than that of the control group,?1d:t=7.227,P=0.0019;2d:t=8.855,P=0.0009;3d:t=15.95,P<0.0001;4d:t=31.83,P<0.0001;5d:t=9.017,P=0.0008?.It indicated that ethionine significantly reduced the proliferation ability of NSCs,and the difference was statistically significant?P<0.05?.5.The flow results showed that compared with the control group,the early apoptosis changed with time,and the apoptosis increased gradually.The late apoptosis also increased gradually.For total apoptosis,apoptosis increased with time,and there were statistical differences between the two groups?total apoptosis 0h vs.72h:P=0.0037,total apoptosis48h vs.72h:P=0.0141,total apoptosis 0h vs.48h:P=0.0084?.6.The expression of Caspase3 protein in the experimental group was higher than that in the control group,p<0.01.7.The flow results showed that the number of cells in the G1 phase of the experimental group was significantly higher than that of the control group?P<0.0001?,while the number of cells in the S and G2 phases was significantly lower than that of the control group(P_S=0.0002,P_G2=0.0021).Statistical significance.The cells of the experimental group were captured in the G1 phase and were unable to enter the S phase.8.The results of immunofluorescence showed that the ability of the experimental group to differentiate into neurons,astrocytes and oligodendrocytes was lower than that of the control group.Conclusions:1.Intraperitoneal injection of ethionine can cause NTDS in C57BL/6 fetal rats,suggesting that methionine circulating metabolic disorder of folic acid is related to NTDS development,and the establishment of this model is a follow-up study on the relationship between methionine circulatory disorder and NTDS.That is,the role of ethionine in NTDS provides an animal model basis.2.Successfully extracted,cultured and identified primary neural stem cells extracted from C57 mice,providing a reliable cell model for subsequent experiments.3.By studying the effect of ethionine on the cell function of neural stem cells,it is preliminarily shown that ethionine may cause the proliferation of neural stem cells,increase the apoptosis and interfere with the differentiation function of neural stem cells in the C57BL/6 mouse embryo.The occurrence of neural tube defects has played a certain role.