【作者】 朱小明；

【导师】 于爱莲； 张忠；

【作者基本信息】 泰山医学院 ， 病原生物学， 2010， 硕士

【摘要】 目的建立脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)致小鼠骨骼畸形的模型；应用双向电泳技术筛选正常胎鼠骨骼与DON所致畸形骨骼的差异蛋白,为进一步阐明DON致骨骼畸形的分子机制奠定基础。方法1、腹腔注射四种剂量DON毒素染毒,获取胎鼠骨骼,骨骼软骨双染色法染色,解剖显微镜观察胎鼠骨骼软骨形态学变化。2、应用研磨法、超声裂解法和裂解液法提取胎鼠椎骨中骨骼与软骨的总蛋白。Bradford蛋白浓度测定试剂盒测定提取蛋白的浓度,确定上样量。3、使用Ettan IPGphor Isoelectric Focusing System和Hoefer SE600分离总蛋白,银染,获取双向电泳凝胶图谱,应用Image Master软件对正常对照组与10mg/kgDON处理组蛋白点间量变变化情况进行分析,筛选差异蛋白。结果1、DON对胎鼠体重和每胎产仔数的影响毒素处理组胎鼠平均体重分别为4 mg/kg组1.10±0.09g、6.4 mg/kg组0.98±0.08g、8.4mg/kg组0.90±0.10 g和10 mg/kg组0.81±0.09 g。毒素处理组平均每胎产仔数分别为4 mg/kg组6.83±3.87只、6.4 mg/kg组6.00±3.63只、8.4 mg/kg组5.00±3.29只和10 mg/kg组5.17±2.71只。实验组胎鼠体重和每胎产仔数与空白对照组相比,差异均有统计学意义,P<0.05。2、DON对胎鼠骨骼软骨的影响实验共出现35种以上骨骼软骨畸形,以中轴骨骼畸形为主,四肢骨骼软骨畸形次之,多发性骨骼畸形多见。其中颈部共出现9种骨骼软骨畸形,以颈椎体融合、颈椎体缺失、颈椎弓融合、颈肋为主。胸骨肋骨共出现6种畸形,包括胸骨缺失、肋骨缺失、肋骨胸骨不对称融合、融合肋、两侧肋骨数目不一、分叉肋,该部位6种畸形均多见。胸椎腰椎共出现15种畸形,以胸椎椎体缺失、腰椎椎弓融合、胸椎椎体碎裂为主。实验还出现3种四肢部位畸形、2种尾部畸形和多种其它部位的畸形。四种剂量中10 mg/kg组引起的畸形种类最多、畸形程度最严重。颈部畸形中10mg/kg组颈椎体融合畸形率、颈椎体缺失畸形率、颈椎弓融合畸形率、颈肋畸形率均高于两对照组,差异有统计学意义,P<0.05。胸骨肋骨畸形中10 mg/kg组胸骨缺失畸形率、肋骨缺失畸形率、融合肋畸形率、两侧肋骨数目不一畸形率、分叉肋畸形率、不对称胸骨连接畸形率均高于两对照组,差异有统计学意义,P<0.05。胸椎腰椎畸形中10 mg/kg组胸椎椎体缺失畸形率和腰椎椎弓融合畸形率均高于两对照组,差异有统计学意义,P<0.05。此外10 mg/kg组六趾畸形率、卷尾畸形率均高于两对照组,差异有统计学意义,P<0.05。3、DON所致畸形骨骼软骨蛋白的双向电泳实验结果成功提取了胎鼠脊椎中骨骼软骨的总蛋白,获得了10mg/kgDON致小鼠骨骼软骨畸形的双向电泳图谱,经Image Master软件分析共筛选到22个差异蛋白点,其中上调蛋白点3个,下调蛋白点19个。结论1、DON可降低孕鼠每胎产仔数和胎鼠体重,具有胚胎毒性；2、DON具有致小鼠骨骼畸形的作用,可诱导小鼠产生多种骨骼软骨畸形,以中轴骨骼畸形为主。腹腔注射10mg/kgDON导致多发性骨骼软骨畸形,可用于制备胎鼠骨骼畸形模型；3、应用双向电泳技术获得DON致小鼠畸形骨骼软骨蛋白的电泳凝胶图谱,筛选出22个差异蛋白点,其中上调蛋白点3个,下调蛋白点19个,为进一步研究DON致骨骼畸形的机制奠定基础。更多还原

【Abstract】 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.更多还原