| Objective:Anorectal Malformations（ARM）are the most common gastrointestinal malformations.The incidence in the world is about 1/50001/1500.Epidemiological and animal experiments show that ARM are complex diseases caused by multiple genes and several environmental factors.Studies on animal models and human specimens show that the occurrence of ARM is closely related to gene mutation,but the major gene has not yet been clarified.In the existing related research,the family of Eph（Erythopoietin producing hepatoma cell line）is involved in cell growth,proliferation,transformation and embryonic development and tumor formation as the largest family of tyrosine protein kinase receptors.In this study,ethylenethiourea（ETU）was used to establish the animal model of ARM by intragastric administration on the 10th day of pregnancy in Wistar rats.Then at the level of genes and proteins,the expression of EphrinB2 in the rectum was detected in normal group and ARM group during the process of cloacal development.The aim of this study was to investigate the spatiotemporal effects of EphrinB2 in the embryogenesis of normal and anorectal malformations.Methods:1.Establishment of animal modelFemale Wistar rats（220-280g）and male Wistar rats（250-300g）were provided by Liaoning Changsheng Biotechnology Company.Male and female adult Wistar rats（1:5）were put in the same cage at midnight and vaginal smears were taken at 8:00 the next day.Pregnancy was defined as the discovery of sperm under microscope and regarded as the first day of pregnancy（E0）.The pregnant Wistar rats were induced by gavage-fed a single dose of 125mg/kg ETU on embryonic day 10 to making ARM rat animal model,the control group received an equal dose of saline.The complete fetus was removed from the cesarean section of rats at E13E16 and divided into normal group and ARM group after deformity was identified.2.Immunohistochemical-paraffin section（IHC-P）Paraffin embedded:Soaked in 4%paraformaldehyde phosphate buffer;dehydrated;embedded in paraffin.Tissue sections:Select the embryo median sagittal continuous slices,slice thickness4μm,the selection can be seen rectal and urethral slide retained.Immunohistochemical-paraffin section（IHC-P）:The sections were gradually dewaxing hydration,antigen heat repair,according to the instructions in turn dropping reagent kit,microscopic observation of DAB and hematoxylin staining,tap water rinse anti blue,gradient alcohol dehydration,xylene transparent,neutral gum seal.3.Microscopic materialEmbryo removed after the cesarean section under a microscope using micro-instruments in sterile conditions Take reproductive nodules placed in the EP tube at-80℃deep frozen,prepared for Western blot and qRT-PCR experiments.4.Western blot and qRT-PCR were used to detect the expression of EphrinB2 at the protein and molecular level.The total protein in the tissue was extracted from the specimens to carry out the quantitative analysis of Western blot.The results of the analysis of the protein content by ProtoBlot II AP system analysis of density,relative gray value represents the protein expression.The other part of the total RNA extracted from the tissue was reverse transcribed into cDNA,the appropriate primer sequence was used to configure the reaction system,and the PCR reaction was performed with an ABI 7500fluorescence quantitative PCR instrument.The relative mRNA level of the target gene was analyzed by 2-△△ct Variety.Explore its expression in the two groups of embryonic distal rectum.5.Statistical AnalysisData 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.ETU made ARM fetal rat animal modelETU teratogenic group,the teratogenic rate of up to 95%or more,generally showed no tail or short-tailed deformity,paraffin sections can be observed ARM fetal rat showed no rectal diaphragma and cloacal membrane fusion,rectal and genitourinary tract There is a fistula,or a permanent cloaca.2.Immunohistochemical staining results EphrinB2 is expressed both during normal and end-stage embryonic mouse embryos in ARM.Normal group:During embryonic development,EphrinB2 expression gradually increased from E13 to E16,showing an upward trend,mainly expressed in the rectal mucosa.ARM group:EphrinB2 expression was stronger in E13E16 than in normal group.3.Western blot and qRT-PCR at the protein and molecular level detection of EphrinB2 expressionThe results of Western blot and qRT-PCR showed that the expression of EphrinB2 in the normal group showed an upward trend with the increase of gestational age,while the expression in the ARM group was higher than that in the normal group.The difference was particularly significant at E13（P<0.05）.At E13,the results of Western blot and qRT-PCR were basically consistent with the trend of immunohistochemistry results.Conclusion:EphrinB2 is expressed in the formation period of anus and rectum,and there is an imbalance in the spatial and temporal expression of EphrinB2.It is believed that EphrinB2 may play a role in the process of cloaca and rectum formation.The upregulation of EphrinB2 at the time of URS division of the cloaca into the primitive recutum and urethra might be associated with the occurrence of ARM. |
