| Objective:Through giving large doses of all trans Retinoic acid (atRA) during pregnancy at one time to establish the model of NTDs in SD rat’s fetal.Through this method, we observ the species、possibility of conggential malformation in experimental group and control group. Record the fetal’s height and weight of the two groups then compare the result.After that,we put the normal and NTDs fetals into pathological sec-tion,observing stracture change and defects.From this we can description the restriction、toxicity and teratogenicity of atRA on embryonic de-velopment.Takeing neural tube tissues on12,13,14,15,16day’s,test the expression of RARaand Hoxd4mRNA to expound the abnormal expre-ssion of RARaand Hoxd4mRNA, provide some theaetical basis at provention、diagnosis and treatment of NTDs.Method:1. Modeling of fetal mouse neural tube malformation:SD mouses which were normally pregnant were randomly divided into two groups, Experimental group were One-time given atRA(120mg/kg) of which concentration is20mg/ml when they were10days of gestation,and olive oil is used for the solvents of atRA. As controls,control group were given the same volume of olive oil when they were10days of gestation. As to get the Fetus,it was needed to profile uterus when they were20days of gestation. Observed the congenital malformations of the embryo, then recorded length, weight and quantity.2. Biopsy:taked the lumbosacral of fetal mouse which was obvious deformity in neural tube in experimental group and which is normal fetal mouse in control group, paraffin-embedded, H-E staining. Pathological changes were observed.3. The detection of RARa and Hoxd4mRNA:Taked the embryonic neural tube tissue from the experimental group and control group at days12,13,14,15and16. RT-PCR semi-quantitative determination was used to detect the expression of RARa and Hoxd4mRNA. Explore the trend of expression during development.Result:1. animal model of neural tube malformation and the malformations:10pregnant mice fetal rat in experimental group accepted statistical at E20-day.The total number of Embryo were109, including stillbirths of5(4.59%),Absorpted fetal of6(5.50%),fetal rats with neural tube defects of65(66.33%),The types of neural tube defects included dominant spina bifida, spina bifida, encephalocele, et.al. all of these were Without deformed tail or anal. Control group which were composed of five pregnant rats, got a total of61fetal mice, wihout stillbirth, fetal absorption, and the appearance of the fetal rat didn’t appeal the above deformity.Experimental group and control group have statistical significant difference between length and weight of each set of data inpregnancy at20days by t-test(P<0.001), The teratogenicity group developed poorlyer than the control group. It was observed in Slice morphological:Control group:sacral, caudal development, spinal cord showed a homogeneous-like, normal morphology, no defects, the boundaries of gray matter are clear, well proportioned.it was observered in the teratogenicity group that obvious malformations of sacrococcygeal structure, dominant spina bifida associated with myelomeningocele, the spinal cord missed or morphological changes of spinal cord, gray matter without normal proportion or the location relationship, the spinal cord was partially exposed, no normal skin wrapped.it was common to see inflammatory cell invasion.2.The reserch about expression of RARmNRA and Hoxd4induced by retinoic acid in the embryos rat neural tube defects:The expression level of RARa in the normal group gradually increased from12days, to peak at14days,since then the expression began to decline, down to a level about that of12days at16days; the expression level of RARa in the control group from12days on was far higher than that of control group,expression levels at16days gradually decreased to just the same as that of the control group at16days,the peak of the expression was advanced, t-test showed significant differences between each group in the control group and experimental group.The expression of Hoxd4in the normal group gradually increased from12days on, to peak at15days, dropped to the level of expression of13days at16days; in control group expression of Hoxd4at12days is far higher than that of in the control group at the same time level, after that the expression decline gradually,To16days, the expression level was slightly lower than the level of the control group at the same time, t-test showed significant differences between each group in the control group and experimental group.Conclusion:1. Added to large dose of all trans retinoic acid at specific time in pregnancy can influence the growth and development of SD rat embryonic, and leaded to varied associated malformations which neural tube defects included.2. The abnormal expression patterns of RARa and Hoxd4gene in SD fetal rat can lead to the occurrence of neural tube defects. |
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