Expressions Of PAX1 And PAX3 Genes In Hindgut Development Of Anorectal Malformation Rats

Objective:In this study,anorectal malformations we investigated Pax1 and Pax3 expressions in normal and in Ethylenethiourea(ETU)-treated embryos development to elucidate its role in the anorectal morphogenesis by immunohistochemistry,Western blot and q RT-PCR technology.Materials:1.Animals: Female Wistar rats(220-250g)and male Wistar rats(250-300g)were provided by Liaoning Changsheng biotechnology company.2.Reagents:(1)Ethylenethiourea,ETU(Sigma-Aldrich Germany)(2)The polyclonal antibody to AB(203065)and AB(180745)were purchased from Abcam,UK;(3)SP secondary antibody kit for immuhistochmistry and DAB reagent were purchased from Mai Xin Bio Ltd.Co.,Beijing,China;(4)GAPDH antiboby were purchased from Proteintech TM;(5)Total protein extraction kit were got from Pik-day Jiangsu Institute of Biotechnology;(6)reverse transcription kit were purchased from Takara bio-Engineering Co.,Ltd.Methods:The pregnant Wistar rats were induced by gavage-fed a single dose of 125mg/kg of 1 % ETU on embryonic day 10 to making ARMs rat animal model,the control group received an equal dose of saline.Respectively,the entire embryos were harvested via cesarean section on E14 to E17.Some of them in anatomical microscope with microscopic instruments under a sterile conditions to take the terminal rectum to EP tube stored at-80 ℃,we evaluated the expressions of PAX1 and PAX3 m RNA and protein by q RT PCR and the western blot.Another embryos were fixed in 4%paraformaldehyde/0.1 M phosphate buffered saline(PBS)for 24 to 36 hours depending on their size.Then the embryos form eatch age group were dehydrated,embedded in paraffin in a routine manner,and sectioned sagittaly at 4μm thickenss.Experiments were carried out as follows:(1)Paraffin embedded tissues were stained with immunohistochemical to observe the development process of the anus and rectum in normal ande ARMs embryo,and the expression S of Pax1 and Pax3 were compared to roughly to position at the intestinal wall in the two groups.(2)Drawn by microscope to obtain the terminal rectum specimens,we evaluated the expression S of Pax1 and Pax3 mRNA and protein by the qRT-PCR and the western blot.The results by Proto Blot II AP analysis system to scan the protein density and analysis to the gradation value represents the relative protein expression.All data were expressed as mean ± standard deviation,compared with the software IBM SPSS Statistics 21,P <0.05 indicates that the difference was statistical significance.Results:IHC-P staining results of Pax1 and Pax3: ARMs were due to the maldevelopment of the URS and its failure to fuse with CM,the anorectum fails to separate from the urethra.A common canal between the retum and urethra still exists,which can lead to a rectoureyhral fistula or persistent cloaca(types of ARMs in ETU-treated rat embryos).During the development of embryonic the expressions of Pax1 and Pax3 were observed in the mucosa and submucosa of the rectum.In Normal Group,As the development of the rectum,the intensity of expression were different.We founs that Pax1 protein expression was significantly in the hindgut on E14 and E15.As the development of the rectum,the expression become less and less..In ARMs Group,We founs that Pax1 protein expression was significantly increase in the ARMs hindgut compared with normal hindgut on E16 and E17.Western blot and q RT-PCR results showed that the expression levels of Pax1 protein and m RNA in the normal group was significantly increase than ARMs(P <0.05).In addition,Pax3 protein expression was significantly increase in the ARMs hindgut compared with normal hindgut on E14 to E17.As the development of the rectum,the expression become more increased.Western blot and q RT-PCR results showed that the expression levels of Pax3 protein and m RNA in ARMs was significantly increase than the normal group(P <0.05).Conclusion:(1)Spatiotemporal expressions of Pax1 and Pax3 was imbalance in fetal period formed cloaca differentiation,anus and rectum formation period,it may play an role;(2)The upregulation S of Pax1 and Pax3 at the time of URS division of the cloaca into the primitive recutum and urethra might be related to ARMs development.