Differential Protein Profiles In 2-DE Of Spinal Cord In Rat Fetus With Spina Bifida And Anorectal Malformation

ObjectiveCongenital anorectal malformation and spina bifida are both common congenital disorders which greatly influence life quality,and incidence are 1‰-5‰,0.2‰-0.7‰, respectively.The causes of these disorders are generally considered as the result from synergistic action of internal agent(genetic factor)and environmental factor (teratogenic factor).However,the aetiology and pathology of these disorders are still unclear,and accordingly no good measures for prevention and therapy.Therefore, studying embryogenesis pattern and early diagnosis of these disorders is necessary to provide message for early treatment and prevention,event-rally to reduce birth defect and raise population diathesis.Though series investigations,we confirmed most cases of congenital anorectal malformation had lumbosacral spinal cord neural developmental anomaly.Among the process of making animal model by medicine's teratogenic effects,we found Retinoid acid and ethylene thiourea both could induce congenital anorectal malformation and spina bifida.At present,we have found Hox gene was involved in the aetiology of these disorders,and SHH,Gli3 were common related genes of these disorders.Several investigations have confirmed folic acid supplementation could prevent the development of neural tube defects,and decrease the number of anorectal malformation and dysmelia.Therefore,we propose a new hypothesis:there may be some common developmental mechanism between congenital anorectal malformation and spina bifida, and spinal nerves maldevelopment may be involved in the development of these two disorders.Proteomics is the science and methodology with which to simultaneously study the expression of all proteins(the protome,rather than individual protein)in a cell, tissue,or organ.Proteomic analysis of identified global proteins can provide expression profiles of the proteins and their PTMs in cell.So,direct investigation on protein profiles is not substitutional.Despite altemative technologies that have emerged, 2-dimensional(2-D)electrophoresis is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures.Furthermore,it delivers a map of intact proteins that reflects changes in protein expression level,isoforms,or post-translational modifications.Last but not least,today's 2-D electrophoresis technology with immobilized pH gradients(IPGs) has overcome the former limitations of carrier-ampholyte-based 2-D electrophoresis with respect to reproducibility,handling,resolution.Here,we employ a 2-D gel-based proteomic technique to detect the global changes of the spinal cord protein expression in rats that have developed animal models, and approach the common mechanism between congenital anorectal malformation and spina bifida at protein level,to provide elementary datas for early diagnosis and treatment during embryonic period.Methods1.Construct rat embryonic models for spina bifida and anorectal malformation.(1)Pregnant Wistar rats were randomly assigned to either control(n=2)or RA (n=19)groups.On E10,RA group received intragastrically all-trans-retinoic acid (160mg/Kg dissolved in olive oil at a concentration of 40mg/ml),whereas control animals received 1 ml of olive oil.(2)On E21,Cesarean section was carried out,and the pups were removed,then spinal cord tissue was removed(form inferior border of the 12^thvertebrae thoracales to the end of spinal cord).2.Differential protein expression 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 IPGphorⅡwas used.Samples were applied to pH 3-10,24cm 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.Results1.Construct the rat model for spina bifida and anorectal malformation.127 rat fetus were obtained totally on E21.20 for control group,and 107 for RA group.Among the RA group,there were 19 fetus for undefect group with RA exposure,29 for isolated anorectal malformation,and 28 for anorectal malformation coincidence with spina bifida in lumbosacral area.2.Analyze the differential protein expression between RA group and control group.The 2D gel protein spot patterns were gained through pH3～10,24cm strips,on average,1355±30 protein spots were detected in control group,and 1425±48,1340±55,1056±68 spots in undefect group with RA exposure,isolated anorectal malformation group and anorectal malformation coincidence with spina bifida group, respectively.In total,we found that 164 protein spots that showed significant differences between the isolated anorectal malformation and anorectal malformation coincidence with spina bifida groups,87 spots from isolated anorectal malformation group were up-regulated,and 77 spots were down-regulated.There were 3 spots which only expressed in isolated anorectal malformation group,and 70 spots in anorectal malformation coincidence with spina bifida group.ConclusionSpinal cord protein profiles of spina bifida and anorecotal malformations were established,and some differential protein spots were found.The data obtained in this study may be helpful for further studies on pathogenesy of spina bifida and anorectal malformation.