The Role Of Vitamin A In The Development Of Enteric Nervous System In Congenital Anorectal Malformation

PART ? THE EXPRESSION OF VITAMIN A IN BLOOD SERUM AND EXPRESSION OF RETINOIC ACID RECEPTORS IN INTESTINES IN CHILDREN WITH CONGENITAL ANORECTAL MALFORMATIONObjective: To investigate the expression profiles of vitamin A(VA)in blood serum and retinoic acid receptors(RARs)in intestines in children with congenital anorectal malformation(ARM).Methods: Serum VA concentrations in newly diagnosed ARM neonates(n=32)and neonates with non-alimentary tract malformations(n=30)were detected.Intestine specimens were divided into 3 groups:rectum of ARM(n=30),colon of stoma(n=30)and rectum of control(n=4),expression of RARs m RNA was evaluated by RT-PCR.Rectum specimens of ARM were divided into 2 groups by postoperative pathology: normal and lesion ganglion cells group,Immunohistochemistry and Western blot were employed to detect the expression of RAR? protein in rectumspecimens.Results: The concentration of serum VA in ARM neonates was lower than in control neonates(P<0.0001).The relative m RNA expression of RAR? was significantly lower in the rectum of ARM compared to the colon of stoma and the rectum of control respectively(P<0.001,P<0.01),but there was no difference between colon of stoma and rectum of control(P > 0.05),no significant difference was found of the relative m RNA expression of RAR? and RAR? among groups(P > 0.05).The RAR?protein were expressed in nucleus of ganglion cells and nerve fibers.The expression of RAR? protein in leision ganglion cells group was significantly lower than that in normal ganglion cells group(P<0.001).Conclusion: There is an abnormal concentration of serum VA and the expression of RAR? in rectum in patients with ARM,which may contribute to the development of enteric nervous system.PART ? THE EXPRESSION OF RETINOIC ACID RECEPTOR ? AND NEUN IN THE RECTUM OF ANORECTAL MALFORMATION MICEObjective: To establish an mouse model of ARM induced by ATRA.To investigate the expression of RAR? and neural marker NeuN in the rectum of ARM mice.Methods: Pregnant mice were randomly divided into six groups(n=5):the group B-D were gavaged 100mg/kg ATRA on gestation day 7.5(GD7.5)and GD8.5 and GD9.5 respectively;the group E-F were gavaged 80 mg/kg and 60 mg/kg ATRA on GD8.5 respectively;the control group A was gavaged same volume of coil oil on GD8.5.The length of fetus body and tail as well as body weight were measured,the number of live,dead and abnormal fetus were recorded on E18.5 respectively.The relative expression of RAR? and NeuN in rectum was evaluated by RT-PCR and Immunofluorescence.Results: The length of fetal body and tail as well as body weight in group B-F were significantly lower than that in group A(P<0.05),but no difference was found among group C,E and F(P > 0.05).The length of fetal body and tail in group C were significantly lower than that in group D(P<0.05).The survival rate of fetus in group B is zero,the incidence of ARMand mortality in group C were significantly higher than that in group D respectively(P<0.05).The incidence of ARM in group E was higher than in group F(P < 0.05),but no significant difference was found between group E and group C(P >0.05).The mortality of group E was lower than that of group C(P<0.05),but there was no significant difference between group E and F.Among the 9 fetuses in experimental groups had neural tube defects(NTDs),the incidence of NTDs in group C was higher than that in group D,E and F respectively(P<0.05),no significant difference was found among group D,E and F(P>0.05).The relative expression of RAR? and NeuN m RNA in ARM fetuses on GD16.5-18.5 was significantly lower than those in control fetuses respectively.The expression of RAR? and NeuN protein was detected in rectum on GD16.5-18.5,the positive staining of RAR? was mainly concentrated in the intestinal muscle region,which partially overlapped with the expression region of NeuN protein.Compared with the control fetuses,the fluorescence intensity RAR? and NeuN were decreased on GD16.5-18.5.Concluion: It is an ideal model for anorectal malformation in C57BL/6J mouse induced by 80mg/kg ATRA on GD8.5.The expression levels of NeuN and RAR? are abnormal in ARM fetuses,excessive ATRA may induce the negative feedback to down-regulation of RAR? in theintestine tissue,thereby affecting the development of ENS in ARM mice.