Relationship Between Retinoic Acid Receptors And Neural Tube Defects Induced By Excess Retinoic Acid

Because of the severely environmental pollution accompanied by the development of the industry, the incidence rate of congenital malformation was rising gradually.More and more scientists began to study the congenital malformation. Neural tube defects was one of the most common and pernicious congenital malformation. Therefore, the study on the morphogenetic mechanism of the NTDs has became popular in teratologic research.The development of neural tube and the establishment of its normal functions were affected and regulated by many factors. Disorders of these factors could induce NTD. It has been proved by a lot of animal experiments that many kinds of teratogens could affect the development of neural tube, and induce a variety of NTDs. Vitamin A and its derivates were the teratogen that were studied mostly. It was proved that excess vitamin A and retinoic acid could induce NTD in mice, golden hamsters and chickens. Although it has been studied on the histologic, cellular, subcellular, even molecular levels, the mechanism of NTD induced by excess vitamin A and its derivates is not yet wellunderstood up to today.Along with the development of molecular biological techniques, it was proved that retinoic acid is a necessary morphogen during embryonic development. It not only determined the establishment of embryonic cephalocaudal axis, but also took a role in the differentiations of the prosencephalon, mesencephalon, epencephalon and spinal cord during the development of central nervous system, and other processes including the migration and fixation of neural crest cells, directional growth of the nervous process. During the development of the brain, it was the nutrition factor related to the differentiation of the neuron, and related to the nerve recovery after nerve injury.The receptor theory is popular for the mechanism of action of retinoic acid. Retinoic acid receptors belong to the superfamily of intranuclear receptors. Mammal retinoic acid nuclear receptors include retinoic acid receptors and retinoid X receptors, and each of them contains three subtypes, α , β and γ. It has been proved that in the initial stage of the embryonic development, there are expressions of various kinds of retinoic acid receptors along anteroposterior axis of the central nervous system, moreover different subtype of the receptors has different expressive pattern. After binding the ligand, RARs and RXRs form dimmer, then bind retinoic acid response elements in the promoters of the target genes, so regulate the transcription of the target genes. It is known that normal embryonic morphogenesis, especially development of the central nervous system is related to the developmental regulation genes regulated by retinoic acid.It is considered that teratogenic effect of excess retinoic acid is probably owing to that retinoic acid unduly activate or inhibit these morphogenetic regulation genes.We have established an animal model of neural tube defects induced by retinoic acid in golden hamster. Furthermore we did some works aboutthe developmental mechanism of NTD on this animal model. In the present experiment, on the animal model, using RT-PCR technique, we investigated the relationship between transcriptions of RAR a and RAR 3 and the neural tube defects induced by retinoic acid;through immunohistochemistry and in situ hybridization techniques, we investigated the differences of RAR a and RARPexpression pattern between the normal genesis and the teratogenesis of neural tube in golden hamster. These researches, not only illuminated the molecular biological mechanism of neural tube defects induced by retinoic acid, but also provided a new experimental evidence for the precaution and therapy for neural tube defects.1. Detection of gene expression involved two levels, mRNA and protein. Methods detecting mRNA include northern blot, RT-PCR and in situ hybridization. First,using RT-PCR technique, we investigated the relationship between transcriptions of RAR a and RAR ￡ and the neural tube defects induced by retinoic acid. Trizol reagent was used to isolate total RNA of neural tube tissues from both RA-treated groups(at 3h,6h, 14h, 22h, 46h and 96h after feeded with RA)and the corresponding control groups respectively. After analysed by electrophoresis and spectrophotometry to evaluate the integrity and purity, RNA samples were reversly transcribed into first strand cDNA with specific downstream primer and reverse transcriptase AMV. Then PCR was performed with specific upstream primer. The PCR products were analysed by electrophoresis on a 2% agarose gel with EB staining. The density of every strap was quantitated with biogel ultraviolet analysator, and the results of treatment and control groups were compared by analysis of variance. We found that expressions of RARa and RAR3 mRNA in the normal embryonic neural tube tissues were time dependant.3 hours after feeded with retinoic acid, expressive level of RARa mRNA was significantly lower than that of the corresponding control group(p < 0. 01);then 6 hours after feeded,expressive level of RARa mRNA was significantly higher than that of the corresponding control group (p < 0.01);14,22,46 hours after feeded, expressive levels of RARa mRNA decreased,but compared with the corresponding control groups, the differences were not significant;96 hours after feeded, its expression increased, even much higher than that of the corresponding control group(/?< 0. 01). 3 and 6 hours after feeded with retinoic acid, expressive levels of RAR3 mRNA were significantly lower than that of the control groups respectively (p< 0. 01, p< 0. 05); 14 hours, expressive level increased, and much higher than that of the control group(p< 0. 01);then 22 hours, expressive level decreased again, and much lower than that of the control group(p< 0. 01) ;46 and 96 hours, expressive levels increased gradually, and the expressive level of 96h was significantly higher than that of the control group(p< 0.01).2. Three types of nucleic acid probes, cDNA, cRNA and oligonucleotide could be used in in situ hybridization histochemistry, and the commonly used marker include two types,the isotope and non-isotope. According to different objective, selected different type of probe labeled with different marker. The RNA probe labeled with digoxin was selected for our present study, because: 1)RNA probe is single strand and does not anneal in the solution, so more probes can hybridize with mRNA in the tissues, thus the signal is stronger. Furthermore the crossbred of cRNA and mRNA is more stable; 2) digoxin is harmless to the health of human and hasn't non-specific stain. We designed specific downstream and upstream primers of RARa and RARP.and the 5' terminal of the primers contains the sites of restriction endonucleases Hindlll and EcoR I . Through RT-PCR technique, RAR a and RAR P fragments were amplified. Then through cutting the purified PCR products and expressive vector pTriEx-4 by restriction endonucleases Hind III and EcoR I , same cohesive terminals were gotten. Using T4 DNA ligase to link target fragment and vector, sodirectional cloning was formed. In the end, through in vitro transcription got the antisense RNA probe labeled with digoxin. The analytic results of restriction endonucleases action and in vitro sequencing confirmed that the sequence of the inserted fragments and the inserted direction were correct, and the specificity of the RNA probes were high, so the probes could be used in in situ hybridization.3. RT-PCR technique detected the changes of the total expressive level of RAR a and RAR 3 mRNA in the tissues of embryonic neural tube. But it is unknown wether expressive levels of RAR a and RAR P mRNA on different sites of neural tube were different. Through in situ hybridization histochemistry and image analysis techniques,we investigated the expressions of RAR a and RAR P mRNA on normal and defected neural tubes of golden hamsters. We found that expressive levels of RARa and RARPmRNA were roughly same on different sites of neural tubes in golden hamster embryos. But in the different developmental periods, expressive levels of RAR a and RAR P mRNA were different. The expressive changes of RARa mRNA in normal groups were that:expressive levels was higher at 3 and 6 hours after feeded with oil, then decreased significantly at 14h, 22h, 46h, at 96h further decreased. The expressive changes of RARP mRNA in normal groups were that:expressive level was the highest at 3 hours after feeded with oil, then decreased significantly at 6h and 14h,at 22h increased again,at 46h and 96h decreased gradually.In experimental groups, 3 hours after feeded with retinoic acid, expressive level of RAR a mRNA was significantly lower than that of the corresponding control group(p< 0.01);then 6 hours after feeded, it was significantly higher than that of the corresponding control group (/?< 0. 01) ; 14, 22, 46 hours after, it decreased, but compared with the corresponding control groups, the differences were not significant;96 hours after, its expression increased, even much higher than that of the correspondingcontrol group(p < 0.01). 3 and 6 hours after feeded with retinoic acid, expressive levels of RARPmRNA were significantly lower than that of the control groups respectively(p < 0.01,/? < 0.05); 14 hours after, expressive level increased, and much higher than that of the control group(p< 0. 01) ;then 22 hours after, it decreased again, and much lower than that of the control group(p < 0. 05); 46 and 96 hours after, expressive levels increased gradually, and the expressive level of 96h was significantly higher than that of the control group (p< 0.05).4. It is the proteins encoded by genes to participate physiological process rather than the genes. Therefore it was not enough only to investigate the expression of RARa and RARP mRNA on normal and defected neural tubes of golden hamsters. The expression of proteins must be studied further. Moreover there was regulatory mechanism during translation of proteins, so expressive levels of protein and gene perhaps were different. Through immunohistochemistry and image analysis techniques, we investigated expressions of RARa and RARP proteins on normal embryonic neural tubes of golden hamsters, and the relationship between expressions of RARa and RARP proteins and NTDs induced by RA.We found that the results were roughly same as those of in situ hybridization histochemistry. It did not suggest that there was not other regulatory mechanism during translation.The results mentioned above suggest:1. the expressions of RARa and RARP on normal embryonic neural tubes of golden hamsters were time dependent. Retinoic acid affects developmental regulatory genes through binding RAR a and RAR P , and regulates the development of central nervous system.2. the expressive changes of RARa and RARP on the defected neural tubes of golden hamsters confirms that RAR a and RAR P take a role in NTDs induced by excess retinoic acid.3. the expressive changes of RARaand RAR 3 on the defected neural tubes of golden hamsters are simultaneous. It suggests that the functions of RAR a and RAR 3 are overlapped, and simultaneous changes of RAR a and RAR 3 can induce NTDs.