Study On The Correlation Of Fibronectin And Laminin ?1 During The Development Of Hindgut With Anorectal Malformation

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TO CONSTRUCT A RAT MODEL OF ANORECTAL MALFORMATIONSObjective: To construct SD rat model of congenital anorectal malformation induced by all trans retinoic acid(ATRA),and to obtain general observation of sacrococcygeal and anal development of fetal rats at different embryonic stages.Detection of the expression levels of Fibronectin and Laminin ?1 during the development of the posterior intestine.Methods: Pregnant female SD rats were randomly divided into control group and model group,with 32 per group.In the model group,ATRA(120mg/kg)was given a gavage at one time at E10.5 days.At the same time,the control group receive the same volume(3.6ml/kg)of simple corn oil by gavage.Fetal rats in both groups were dissected on embryonic period of E11.5,E12.5,E13.5,E14.5,E15.5,E16.5,E18.5,and E20.5,and the number of fetal rats was recorded.Gross images of embryos were collected at each time point,weight,body length,tail length of fetal rats in both groups were measured,and the incidence of congenital anorectal malformation was collected.Results: In control group,378 fetal rats were obtained,all of these embryos hadn't be observed with anorectal malformation.In model group,361 fetal rats were collected,and the incidence of anorectal malformation was99.4%(359/361).HE staining results showed that the rectum communicated with the outside at E16.5 in control group,forming an anal opening,while the rectum in model group did not communicate with the outside,forming a blind end or communicating with the ventral urogenital system by the form of fistula.The general image of the embryos showed that,comparing with the control group,the model group demonstrated abnormal sacrococcygeal development at each development time point,with no tail(292/361)or short tail(69/361).At E18.5 and E20.5,the fetal rats also showed no anal opening except short tail or no tail.The weight,body length,and tail length of the fetal rats in the model group lower than that of the control group at each development time point,and the differences were more significant after E16.5.Through independent sample t test and U test suggested that the differences were statistically significant(P<0.05).Conclusion: Large dose of ATRA was gavaged at one time during pregnancy could successfully induce the congenital anorectal malformationsof SD embryos which is similar to the pathyoloy of human's anorectal malformation,and this settef up a foundation for subsequent experiments.PART ? EXPLORATION THE EXPRESSION AND SIGNIFICANCE OF FIBRONECTIN & LAMININ?1DURING THE DEVELOPMENT OF HINDGUT OF FETAL RATS WITH CONGENITALANORECTAL MALFORMATIONObjective: To investigate the expression level and significance of Fibronectin(FN1)& Laminin?1(LAMB1)during the development of hindgut of fetal rats with anorectal malformation model(n=32)in SD rats.Methods: Embryos were dissected of both control group and model group at E11.5-E16.5,E18.5 and E20.5 to collect cloacal/rectal specimens,at the same time,the serum of pregnant rats was collected.q RT-PCR was used to detect the expression of Fn1 and Lamb1 m RNA between control group and model group.The expressions of FN1 and LAMB1 proteins were further detected by Western blot and Immunohistochemistry.Results: Fn1 and Lamb1 were continuously and dynamically expressed in the development of hindgut.In control group,q RT-PCR results showed that the m RNA of Fn1 and Lamb1 gradually increased from E11.5,and the expression level reached the peak at E15.5,after then gradually decreased.In model group,at each time point(except for Lamb1 at E20.5),the expression of both gene were significantly higher than that of the control group,with significant difference statistically(P < 0.05);they began to rise from E11.5until E16.5,reaching the peak during the whole development of hindgut,then fell sharply,to E20.5,Fn1 has dropped significantly but its expression level was still higher than control group,while the Lamb1 fell to the same level of control group.The results of Western blot showed the same trend as the q RT-PCR,and the difference of protein expression level between control group and model group was statistically significant(P<0.05).In control group,the expression levels of FN1 and LAMB1 reached the peak at E15.5 during the development of hindgut.However,in model group,except the expression level was up-regulated,the time of peak also delayed,which appeared at E16.5 days.Immunohistochemistry results demonstrateded that,in control group,FN1 and LAMB1 were mainly uniformly distributed in the mucosal layer and basement membrane of the cloacal epithelium,and the change trend of expression level was consistent with the results of Western blot and q RT-PCR;in model group,the expression level of FN1 and LAMB1 were not only up-graduated,but also uneven distribution.Conclusion: Compared with control group,the expression levels of m RNA and protein of Fn1 and Lamb1 were observably up-regulated,the time of peak were delayed,and the distribution were disordered in model group during the development of hindgut.these changes may disrupted the occurrence of normal biological events in the cloaca development and which maybe relate to the occurrence of Anorectal malformation.