| Objectives To investigate the influence of growth and development about all-transretinoic acid-induced neural tube defects SD fetal, Analysis of the types of abnormalities andits ratio, the change of spinal cord neural stem cells biological characteristics and H3K27me3.Research folic acid for its role in the relationship on the intervention. Explore the mechanismof neural tube defects and folic acid preventive effect, providing a more reliable theoreticalbasis for the prevention of neural tube defects.Methods1. Establish animal model: To randomly divided the sexually mature femalerats into①normal control group,②teratogenic model group,③folic acid interventiongroup.Two weeks before the start of③group rats mating fed diets containing folic acid foodto cesarean births;②and③groups were One-time given atRA (120mg/kg) of whichconcentration is40mg/ml when they were10days of gestation, and olive oil is used for thesolvents of atRA. as controls, control group were given the same volume of olive oil whenthey were10days of gestation. To get the Fetus,it was needed to profile uterus when theywere20days of gestation. Observed the fetal gross morphology, then recorded quantity,weight and length.2. Pathological observation: Take the same significant site of fetal neural tube defectsand normal fetal tissues, paraffin-embedded HE staining, pathological changes were observedunder a microscope.3. Spinal cord neural stem cell isolation and culture: Take three groups E15d embryonicspinal cord, which were isolated and cultured neural stem cells, then statistics the changes ofthe NSCs ratio、growth activity and differentiation characteristics in each group.4. Detect H3K27me3expression in each group Neural stem cells: Qualitative judgmentsH3K27me3expression in three groups by the immune cytology and quantitative analysis ofH3K27me3expression of the nucleus in the three groups by quantitative Western blottechnique.Results1.①normal control group get received a total of117fetuses, one stillbirths,therest were live births, look no obvious deformity;②teratogenic model group stillbirth or fetal absorption12,93live births, dominant spina bifida45.2%(42/93), no tail, ring-tailed macaquedeformity66.7%(62/93), foot deformities22.6%(21/93), anencephaly and encephaloceleand other deformities8.6%(8/93);③folic acid intervention group Stillbirth or fetalabsorption7,106live births, the dominant spina bifida26.4%(28/106), no tail or ring-tailedmacaque deformity42.5%(45/106), foot deformities11.3%(12/106), anencephaly andencephalocele malformations5.7%(6/106). The control group received a total of125fetuses,②compared height and weight with①were poorly developed, t-test analysis, thedifference was statistically significant(P <0.05),③group developed better than①group, thedifference was statistically significant (P <0.05).Pathology observed: Normal fetal rat spinal cord have a normal morphology, vertebraland cone arch well developed, continuous coating of skin, fetal neural tube mainly closurewhen E15d, central canal morphology rules. Dominant spina bifida fetal rat spinal cord tochange complex (high position compared with the folic acid intervention group), spinaldorsal skin covered with discontinuous tissue defects, along the longitudinal axis of the spineshowed an oval defect area, about0.3~0.4cm(range was larger compared with folic acidintervention group), deep spinal cord tissue loss, morphological changed, spina bifidaoutward, multi inflammatory cell infiltration, the end of caudal could see full disordermulti-polar neural epithelium arranged to form a plurality of tubular structures to form neuraltissue hamartoma tumors.3. Immunocytochemistry display the cells obtained Nestin positive, and could beinduced differentiation of GFAP, NSE positive cells，with NSCs characteristics.①normalcontrol group,②teratogenic model group,③folic acid intervention group Microscopic nerveball counts were as follows:29.4±2.07、17.8±2.17、23.4±1.52（P<0.05）;diameter:140.2±4.82、106.6±3.85、121.8±4.09（P<0.05）;Nestin expression levels after induction ofdifferentiation were:（6±0.9）%、（21±4）%、（32±3）%（P<0.05）.Immunocytochemistryrevealed three groups of neural stem cells are all express H3K27me3ion, Western blotanalysis showed abnormal group H3K27me3expression was significantly higher than thenormal group, folic acid intervention group down regulated (P <0.05) than the teratogenicmodel group.Conclusions1. All-trans retinoic acid can induce a variety of neural tube defects;2. Neural tube defects growth retardation, folic acid can improve this phenomenon;3. Serum-free culture conditions for spinal cord stem cells grew well, maintaining the characteristics of stem cells;4.Neural stem cells in a lower proportion of neural tube defects, and the proliferationand differentiation capacity is weak, folic acid interventions can play a protective role;5. Folic acid to adjust the three methylation status of H3K27in neural stem cell nuclei,resulted in changes about the biological characteristics of neural stem cells. |
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