Study About The Intervention Effect Of Folic Acid On Pax3 And H3K27me3 In NTDs Rats

Objectives: With modeling on SD-strain rats with NTDs(Neural Tube Defects) induced by ATRA(All-trans Retinoic Acid), this study devoted understanding about the Pax3’s expression and the level of H3k27me3 during the formation of neural tube closure defects, then meanwhile confirmed the folic acid’ s effect on such phenomenon, and further explored the folic acid intervention’s effect on prevention of NTDs and its possible generating mechanism, so as to provide reliable theoretical reference for the prevention of this congenital defects.Methods: 1. Establishing of experimental animal model:divided the sexually mature healthy female rats into three groups randomly: normal control group, teratogenic model group, folic acid- intervention group. Feeding the third group with folate(60mg/kg) two weeks before pregnancy until to the end of gestation; And ATRA at 100mg/kg was once given by gavage to both folic acid- intervention and teratogenic model groups on gestational days 10, while equivalent volume of olive oil was administered by intragastric infusion for the normal control rats.2. Morphological observation: caesarean on each samples of each group were operated on gestational days 15 to observe the pathological phenotype, and counting the embryon consisting of living embryons, lethal foetus, absorved embryons, defective individuals and nomal embryos, which in order to make statistical analasis about the incidence of malformation.3. Pathological observation: before abserved the pathogical changes by using microscope, following operations were conducted: embryos fixation and dehydrated them through gradient varing concentration of ethanol; then put them into dimethrlbenzene solusion to make transparent. After all of these steps, embed paraffin into the embryos, sliced conven-tionally and then stained with Hemataxylin and Eosin(H.E).4. Determination of folic acid and homocysteine(Hcy) in blood serum: adopted intraperitoneal anesthetic injection to make the pregnant maternal rats numb. After collecting cardiac blood from heart in thoracic cavity whose external furriery was disinfected commonly into centrifugal tube for about 30 minutes, diluted the supernatant collected from the centrifuged blood at 3000 rpm by phosphate buffer at a percentage of 10:1, and then put them into EP tubes respectively to be detected.5. Determination of Pax3 genic expression: immunohistochemical techniques were used to carry on qualitative analysis of Pax3 genic expression indirectly of embryonic tissue, while the quantitative analysis was conducted by western bloting and QRT-PCR techniques.6. H3K27me3 detection: make qualitative analysis on the levels of H3k27 me of each embryonic tissue by using immunohistochemical and western bloting techniques.Results: 1. The results of the counting of defective embryo showed that no malformations exist in the control group, while 98.61% and 35.09% incidence of defects respectively appeared in the teratogenic model and folic acid-intervention groups. Of the normal control group’s, there were 60 living embryons, 1 lethal and 61 normal phenotype, absorved embryons and deformitise were undiscovered. The uterine malformation of pregnancies was in account of dead fetus, of which, NTDs was exclusided. Of the defective group’s, there were 72 living embryons, including 71 defects and 1 normal embryos. 57 living embryos were absorved in the folic acid-intervention group, among of which were 37 normal embryos and 20 malformations, the absorved were undiscovered.2. Pathological observation revealed that upgrowth of normal control group’s embryos was normal, and the layers of organization structure were clearly visible. The dorsal skins and subcutaneous tissue of teratogenic model group’s embryos which had dominant rachischisis appear oval coloboma, the structure deep in the spinal cord were disorders and swell outside, which also often accompanied with inflammatory cells’ infiltration. Besides, compared to former one, the rachischisis’ position of embryos in folic acid-intervention group was lower, so the proportion of coloboma and damage extent was reversed.3. Folic acid in pregnant maternal rats’ blood serum of three groups were respectively(62.350±10.678)ng/ml,(46.650±6.143)ng/ml and(117.375±6.564)ng/ml, the Hcy in blood serum were respectively(2.875±0.419)μmol/l,(4.825±0.448)μmol/l,(3.500±0.265)μmol/l.4. Immunohistochemical techniques in this study showed that there were 2(-)samples, 10(+) in normal group; 2(++)and 11(+++) appeared in teratogenic model group; 9(++)and 3(+++)expressed in folic acid- intervention group.H3K27me3 in normal group was slightly expressed,while stronly positive expression existed in teratogenic model(p<0.05).Compared with teratogenic group,H3K27me3 expressed in acid- intervention model was lower,meanwhile compared with control group,it’s expression had apparent significance(p<0.05).5. The results of Western bloting revealed that the expression of Pax3 was significantly lower when compared to control group;On the contrary,the expression of Pax3 in Folic acid-intervention group was increased and restored to normal status.In addition,the level of H3k27 trplemethylation had a negative correlation with Pax3’expression The result of expression of Pax3 detected by QRT-PCR was consistented with the result by Western bloting.Conclusion: 1. Folic acid can effectively reduce the incidence of NTDs induced by All-trans retinoic acid;2. Pax3 genic expression of rats with NTDs reduces, while the level of H3k27me3 rises, and folic acid- intervention can effectively correct this phenomenon;3. Very tight connection exists among the occurrence of NTDs,folic acid protection and Pax3, H3k27me3.