| Objective: The aim of this study was to determine the expression of TBX4(T-box transcription factor 4)during anorectal development in normal and ethylenethiourea(ETU)-induced anorectal malformation(ARM)embryos with a view to establishing the possible role of TBX4 in ARM pathogenesis.Methods: The Wistar rats were mated for the night,the next morning the female rats were given vaginal smear.When the sperm was seen under the microscope,it was suggested that the rats had mated,the day was marked as gestational day 0(E0).The pregnant rats were randomly divided into two groups,ETU-treated group and control group.In the ETU-treated group,pregnant rats were given by gavage with 1% ETU solution in dose of 12.5m L/kg on E10.The control rats were given by gavage with normal saline in the corresponding dose on E10.All the rats were continued to maintain pregnancy.The ETU-treated group and the control group rats were respectively to subject the following process: From E13 to E16,the embryos were removed from the pregnant rats.The pregnant rats were treated with 10% hydrochloral(4m L/kg)by intraperitoneal injection.All the fetal rats were removed and the whole embryos were taken.All specimens were rinsed with 0.01 M PBS.A part of embryos were fixed in 4% paraformaledehyde phosphate buffer for 72 hours,dehydrating,embedded in paraffin.The wax blocks were continuously sliced in the middle sagittal plane,and the slice thickness was 4μm.The sections were divided into normal group and ARM group.The immunohistochemical staining was performed on these sections.The rest of embryos were divided into normal group and ARM group under the microscope.The expression of TBX4 protein in normal and ARM groups were detected by Western Blot,and the expression of TBX4 m RNA in normal group and ARM group was detected by RT-PCR.Results: Immunohistochemical results showed that on E13,TBX4-immunopositive cells were distributed in the epithelium of hindgut and urogenital sinus.On E14,TBX4-immunopositive cells began to appear at the cloacal canal and the epithelium of hindgut in the normal rat embryos.A large number of TBX4-immunopositive cells were observed in the future anal orifice part of cloacal membrane.At the same time,TBX4-immunopositive cells were seen in the dorsal epithelium of the urogenital sinus.On E15,the epithelium of the rectum and the anal membrane were immunopositive for TBX4.On E16,TBX4-immunopositive cells were also detected in the epithelium of terminal rectum and the dorsal side of the urethra,but the number was significantly lower than that of E15.ARM group: On E13,no TBX4-immunopositive cells were seen in the hindgut or cloacal membrane.On E14,the positive cells scattered in the cloacal canal.Only a small number of positive cells were found in the hindgut near the cloacal canal.There are only a few of positive cells in the most dorsal part of the cloacal membrane.On E15,a fistula is seen between the rectum and urethra.The expression of TBX4 in hindgut epithelium,fistula,urethral epithelium and the most dorsal part of the cloacal membrane was very weak.The specific positive staining was not obvious.On E16,positive cells scattered in the fistula epithelium.There were only sporadic positive cells in the epithelium of the distal rectum and the dorsal side of the urethra.Western Blot results showed that TBX4 protein was expressed in both normal and ARM groups from E13 to E16.From E14 to E16,the expression of TBX4 in ARM group was lower than that in normal group.On E15,the expression of TBX4 protein in ARM group was significantly lower than that in normal group.(E15: TBX4 protein: 1.25±0.67 vs 2.13±1.01,P<0.05).RT-PCR results showed: From E13 to E16,the expression of TBX4 m RNA gradually increased in the normal embryo.On E13 and E15,the expression of TBX4 m RNA significantly decreased in the rectum of ARM rat embryos comparing with normal rat embryos(E13 TBX4 m RNA: 0.48±0.18 vs 1.20±0.62,P<0.05;E15 TBX4 m RNA: 0.96±0.35 vs 1.61±0.44,P<0.05).Conclusions: The expression of TBX4 were downregulated in anorectal unit of fetal rats with anorectal malformations induced by ethylenethiourea,which may play important roles in the pathogenesis of anorectum development. |
