The Model Of Skeletal And Cartilage Malformation Induced By The Intraperitoneal Administration Of Deoxynivalenol In Mice And The Screening Of Relating Distinct Proteins

ObjectiveThe aims of this article are as the following:1 to establish the model of skeletal deformity induced by DON in mice; 2 to find distinct proteins between normal mice and skeletal malformed ones by two-dimensional gel electrophoresis for further study of the mechanism of teratogenicity of DON.MethodsMice were treated by intraperitoneal administration of deoxynivalenol and then the skeletal and cartilage of mice were obtained to observate the morphological changes by dissecting microscope after double-staining according the control ones. Finally the total proteins of abnormal skeleton and cartilage of vertebrae in fetal mice were extracted applying grinding, ultrasonic decompose, lysis buffer and other necessary solutions, and the concentration of extracted protein were detected by Bradford protein assay kit to determine protein loading amount, and they were separated applying Ettan IPGphor Isoelectric Focusing System according to the differences of their isoelectric point (PI) by isoelectric focusing electrophoresis, then the isolated proteins were further separated applying Hoefer SE600 basing on their molecular weight by SDS-polyacrylamide gel electrophoresis. At last the Silver staining was carried out to obtain the 2-DE illustration, and the quantitative changes between the treated samples and the control ones were scanned and analyzed to find potential distinct proteins affected by DON with Image Master software.Results(1) Effect of DON on the weight of fetal and the number of offspring in one litterThe fetal weight of four treated groups were 1.10±0.09 g,0.98±0.08 g,0.90±0.10 g and 0.81±0.09 g, respectively. The number of offspring per litter of them were 6.83±3.87 rats,6.00±3.63 rats,5.00±3.29 rats and 5.17±2.71 rats. Compared with that of the control group, the differences of average fetal weight and the offspring amount per litter were statistically significant, P<0.05.(2) Effect of DON on skeleton and cartilageMore than thirty-five skeletal deformities were observed including primary axial skeletal deformity, secondary limb deformity, and multiple deformity was common too.Nine skeletal deformities were observed in neck such as cervical fusion, cervical deletion, cervical arch fusion and vertebral rib. There were six costal and sternal skeleton deformities as these:sternal deletion, costal deletion, asymmetric sternocostal junction, fused ribs, costal asymmetry and branched ribs. There were fifteen thoracolumbar vertebral skeletal deformities such as thoracic vertebral arch fusion, thoracic vertebrae deletion, thoracic vertebrae fragmentation and so on. There were three kinds of deformities in limb, two in tail and many in other parts.There were remarkable differences in cervical detected deformity rate between10 mg/kg group and the two control groups, P<0.05. So were they of cervical fusional deformity rate, cervical arch fusional deformity rate and vertebral rib deformity rate between10 mg/kg group and the two control groups, P<0.05.There were remarkable differences in sternal deletion deformity rate between10 mg/kg group and the two control groups, P<0.05. So were they of costal deletion deformity rate, fused costal deformity rate, costal asymmetric deformity rate, branched costal deformity rate and asymmetric sternocostal junction rate betweenlO mg/kg group and the two control groups, P<0.05.There were remarkable differences in thoracic vertebrae deletion rate between10 mg/kg group and the two control groups, P<0.05. So was it of lumbar vertebra arch fusional deformity rate between10 mg/kg group and the two control groups, P<0.05.There were remarkable differences in hexadactylism deformity rate between 10 mg/kg group and the two control groups, P<0.05. There were remarkable differences in coil tail deformity rate betweenl 0 mg/kg group and the two control groups, P<0.05.(3) The result of 2-dimension gel electrophoresis for the skeletal abnormalities induced by DON in miceTotal proteins were successfully extracted from fetal spine of skeletal deformity and 2-dimension electrophoresis illustration were obtained.22 significant distinct proteins were primarily screened including 3 up-regulated proteins and 19 down-regulated ones analyzed with Image Master.Conclusion(1) DON can reduce the number of litter size and fetal body weight, i.e. DON posses embryo toxicity in mice at least.(2) DON has the capacity of inducing skeletal deformities. The majorities of those deformities belong to axial skeleton. The dose of intraperitoneally injected 10mg/kg induced the most serious and species skeletal deformities, which were almost multiple malformations.10mg/kg was the preferred dosage for inducing fetal malformation model.(3) 2-dimension electrophoresis of skeleton and cartilage deformities induced by DON were obtained, and 22 distinct deformity relating proteins were primarily screened including 3 up-regulated proteins and 19 down-regulated ones, which will be the foundation for further research on the mechanism of teratogenicity of DON.