Spatiotemporal Expression Of PPPDE1 In The Development Of Anorectal Malformations In Fetal Rats

Objective: To determine the expression of PPPDE1 gene during anorectal development in normal fetal rats and malformation(ARM)fetal rats,and to analyze the pathogenesis of the gene in the development of anorectal malformations in rats.Methods:Healthy rats were purchased,female and male rats were caged midnight and smeared in the morning,confirming that pregnant rats were cage-separated.The pregnant rats were divided into two groups: a normal group and an ethylenethiourea(ETU)-treated group.The 36 pregnant rats were gavage-fed a single dose of 125mg/kg of 1% ETU on embryonic day 10(E10=sperm in vaginal smear after overnight mating).The 30 pregnant rats in the normal group were treated with the same dose of12.5ml/kg saline on E10.Embryos were removed from E13 days to E16 days of pregnant rats.Approximately a third of the embryos were fixed in 4%paraformaldehyde for 12-24 hours.The rats tissue specimen were subjected to a conventional dehydration treatment and embedded in paraffin.And teated to serial sagittal sections were cut at 3.5?m thickness for immunohistochemical staining(IHC)with PPPDE1 genes.The other specimens were taken under the low temperature conditions to take out the rectum tissue of pregnant rats and stored in the refrigerator at-80 centigrade.The specimens were prepared for western blot and q RT-PCR.Statistical Analysis: Data were expressed as mean ± standard deviation,differences were compared using IBM SPSS Statistics 21 statistical software for t test to P value that statistical differences,p<0.05 that the difference was statistically significant.Results: 1.Immunohistochemical staining results,in normal embryos,PPPDE1 immunopositive cells were sparsed detected in the URS and CM on E13.PPPDE1 immunopositive cells were detected in the URS,hindgut and CM on E14.PPPDE1 immunopositive cells were observed the fused tissue of the URS and the thin anal membrane on GD15.On E16,PPPDE1 immunolabeled cells were observed on the anal epithelium.In ARMs embryos,on E13,PPPDE1 mmunopositive cells were extensively detected in the URS,hindgut epithelium.PPPDE1 mmunopositive cells were significantly increased in the URS,hindgut on E14.PPPDE1 mmunopositive cells were distributed in the fistula and hindgut epithelium on E15.The fistula and rectal epithelium with PPPDE1 mmunopositive cells on E16.2.The results of Western blot,the expression of PPPDE1 protein changes rely on temporal change in hindgut development.The expression of PPPDE1 protein in E13-E14 ARMs group was significantly higher than that in normal group(E13,0.54 ± 0.03(normal group)vs.0.67 ± 0.08(ARM group);E14,0.48 ± 0.003(normal group)vs.0.65 ± 0.11(ARMs group);p <0.05),the difference was statistically significant.3.The results of q RT-PCR,PPPDE1 m RNA expression was significantly decreased in the ARMs fetuses compared with normal fetuses on E13 and E16.Compared with normal fetuses,ARMs group had a significant difference on E13(E13,1.00±0.04(normal group)vs.1.24±0.13(ARMs group);p <0.05)the difference was statistically significant.Conclusion:1.In ARMs embryos,the expression of PPPDE1 was characterized by time-space imbalance of fetal rats with teratogenic deformities.2.The expression pattern of PPPDE1 during embryonic development of rat embryonic teratogenesis may cause the ARMs embryos,affecting the apoptosis of cloaca cells.3.Increased PPPDE1 expression might be related to the development of ARMs.