| ObjectiveCongenital anorectal malformation and spina bifida are both common congenital disorders, and incidence are 1‰-5‰,0.2%o-0.7%o. It is generaly known that congenital norectal malformation and spina bifida are both polygenic inheritance.These disorders are caused by a combination of genetic and environmental factors. However, the aetiology and pathology of these disorders are still unclear, and accordingly no good measures for prevention and therapy. Therefore, studying embryogenesis of these disorders is necessary to provide message for early treatment and prevention.Our revious studies of pathohistology, nerve tract tracing, neuroelectrophysiology and X-ray, we found the similar abnormalities of spinal cord in both congenital anorectal malformation and spina bifida. Meanwhile, retinoid acid and ethylene thiourea could induce both congenital anorectal malformation and spina bifida in animal models. Furthermore, many genes (Hox, SHH and Gli3) were involved in both disorders. Several investigations have confirmed folic acid supplementation in pregnant women could decrease the incidence neural tube defects and anorectal malformation. Therefore, we propose a new hypothesis:spinal nerves maldevelopment may be involved in the development of both congenital malformations.Proteomics is the methodology with which to simultaneously study the expression of all proteins in cell, tissue, or organ. Proteomic analysis of identified global proteins can provide expression profiles of the proteins and their PTMs in cell. Proteomics characterized by 2-DE and mass spectrometry has attracted much attention in recent years. Since it is difficult to perform biopsy for spinal cord in human subjects,it is necessary to study the pathogenesy of the congenital anorectal malformation and spina bifida with the rat model. In present study, we screened and identified many proteins involved in embryogenesis of both congenital anorectal malformation and spina bifida using 2DE and mass spectrometry.Methods1. Rat embryonic models for spina bifida and anorectal malformation.(1) Pregnant Wistar rats were randomly assigned to either control (n=9) or RA (n=24) groups. On E10, RA group received intragastrically all-trans-retinoic acid (135mg/Kg dissolved in olive oil at a concentration of 40mg/ml), whereas control animals received 1ml of olive oil.(2) On E17, rat fetus were removed through laparotomy, then spinal cord tissue was dissected (form inferior border of the 12th vertebrae thoracales to the end of spinal cord)2. proteomic analysis of spinal cord between control group and RA group in the rat fetus.(1) Total protein of spinal cord was extracted from control and RA rat fetus, protein concentrations were determined by Bradford.(2) For isoelectric focusing, Ettan IPGphor II was used. Samples were applied to pH 4-7,24cm non-linear IPG strips and two-dimensional electrophoresis was performed, using 12.5% second dimension gels. Analytical gels were silver stained and scanned. Protein patterns were analyzed using ImageMaster 2D Platinum 6.0 software to find differential spots.(3)Significant protein spots were selected for mass spectrometry analysis and bioinformatics analysis.3. The expression of Erp57 in control group and RA group in the rat fetus(1) Proteins are extracted from myeloid tissue of rat fetus, quanitated by Bradford.(2)The expression of Erp57 in spinal cord is demonstrated by Western-blot,which helps us to study the relation with diseases.Results1. Rat model for spina bifida and anorectal malformation.358 rat fetus were obtained totally on E17.100 for control group,and 231 for RA group. Among the RA group,there werel fetus for undefect group with RA exposure,79 for isolated anorectal malformation, and 127 for anorectal malformation coincidence with spina bifida in lumbosacral area. The other including clubfoot coupled with spina bifida and imperforate anus, gastroschisis. And incidence of absorption rate was 8.1 %(21/258), incidence of dead rate was 2.8%(6/258).2. Separation of proteins.The 2D gel protein spot patterns were gained through pH4-7,24cm strips, with good reproducibility and resolution.On average,825±30protein spots were detected in control group,and 820±30,800±70 spots inisolated anorectal malformation group and anorectal malformation coincidence with spina bifida group, respectively. In total, we found that20 protein spots that showed significant differences between the normal and the disease groups, compared with the contral group 10 spots were up-regulated, and 7 spots were down-regulated from the disease groups,。3. Identification of proteins.20 easily to picked sports that have good reproducibility and obvious differences are charactered by MS,19 sports were identified,one of them can not be finded in the number library.At last,13 strains proteins were found have similar expression in the both desease groups,, compared with the contral group 6 strains were up-regulated, and 7 strains proteins were down-regulated. Western-blot results find the expression of Erp57 is up-regulated in the both disease group,which is coincidence with the result of 2DE.ConclusionThe proteomic analysis of spinal cord in rat fetus with spina bifida and anorecotal malformations were established,and some differential protein spots were found.Proteomic difference between disease fetus and normal contrals was presented,and the alterration of the 13 differentially expressed proteins such as Erp57 are found.
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