脱氧雪腐镰刀菌烯醇致小鼠骨骼畸形模型建立及差异蛋白的筛选
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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。
Objective
The 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.
Methods
Mice 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 litter
The 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 cartilage
More 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 mice
Total 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.
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。
Objective
The 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.
Methods
Mice 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 litter
The 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 cartilage
More 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 mice
Total 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.
引文
[1]霍兴华,赵宝玉,万学攀,等.脱氧雪腐镰刀菌烯醇的毒性研究进展[J].毒理学杂志,2008,22(2)：151-154.
[2]Pestka JJ.Smolinski AT.Deoxynivalenol:toxicology and potential effects on humans [J].J.Toxicol.Enyiron.Health B Crit.Rev,2005,8(1):39-69.
[3]Centin Y,Bullerman LB.Cytotoxicity of Fusarium mycotoxins to mammalian cell cultures as determined by the MTT bioassay[J].Food Chem Toxicol,2005,43:754-764.
[4]Bimezok D.Doll S.Rau H, et al.The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cell in vivo and in vitro [J].Immunobiology,2007,212:655-666.
[5]Peska JJ,Amuzie CJ.Tissue distribution and proinflammatory cytokine gene expression following acute oral exposure to deoxynivalenol:Comparison of weanling and adult mice[J].Food and Chemical Toxicology,2008,46:2826-2831.
[6]Doll S,Schrickxa JA,Danickeb S, et al.Deoxynivalenol-induced cytotoxicity, cytokins and related genes in unstimulated or lipopolysaccharide stimula-ted primary porcine macrophages[J].Toxicology Letter,2009,284:97-106.
[7]李群伟,侯海峰,李亚鲁,等.脱氧雪腐镰刀菌烯醇致家兔多器官损伤的实验观察[J].中国地方病防治杂志,2007,22(1)：19-22.
[8]Konigs M,Lencxyk M,Schwerdt G, et al.Cytotoxicity,metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture[J].Toxicology,2007,240:48-59.
[9]Yang H,Chung D H,KimY B, et al.Ribotoxic mycotoxin deoxynivalenol induces G2/M cell cycle arrest via p21Cip/WAFI mRNA stabilization in human epithelial cells[J].Toxicology,2009,243:145-154.
[10]Bony S,Carcelen M,OlivierL, et al.Genotoxicity assessment of deoxynival-enol in the Caco-2 cell line model using the Comet assay[J].Toxicology Letter,2006,166:67-76.
[11]Manda G,Neagu M,Constantin C, et al.Immunotoxicology of mycotoxins produced by Fusarium fungi-Low concentrations of deoxynivalenol interfere with nucleotide metabolism[J].Toxicology Letter,2007,172:S49.
[12]王加生,徐达道.赤霉病麦中毒研究ⅣV.赤霉病麦粗毒素的致畸与致突变研究[J].真菌学报,1986,5(1)：52-62.
[13]庞炜,王志伦,吕社民,等.真菌毒素DON研究进展[J].中国地方病防治杂志,1996,11 (6)：349-351.
[14]Vesely D, Vesela D. Embryo toxic effects of a combination zear-alenone and vomitoxin(4-dioxynivalenone) on the chick embryl [J].Vet Med Phaha,1995, 40(9):279-281.
[15]Morrissey RE.Norred WP.Vesonder RF.Subchronic toxicity of vomitoxin in Sprague-Dawley rats[J].Food Chem Toxicol,1985,23(11):995-999.
[16]Debouck.C.,Haubruge.E, Bollaerts.P, et al.Skeletal deformities induced by the intrapertoneal administration of deoxynivalenol (vomitoxin) in mice [J].International Orthopaedics,2001,25(3):194-198.
[17]Collins TF,Sprando RL.Black TN, et al. Effects of deoxynivalenol (DON, vomitoxin) on in utero development in rats[J].Food Chem Toxicol,2006,44(6):747-757.
[18]Khera KS, Whalen C, Angers G, et al. Embryotoxicity of 4-Deoxynivalenol (Vomitoxin) in Mice[J].Bull Environm Contain Toxicol.1982,29 (4):487-491.
[19]李勇,王维林,袁正伟,等.一种简捷可靠的胎鼠骨和软骨双重染色技术[J].中国医科大学学报,2004,33(2)：189-190.
[20]Young AD, Phipps DE, Astroff AB. Large2scale double2staining of rat fetal skeletons using Alizarin Red S and Alcian Blue [J].Teratology,2000,61 (4):273-276.
[21]郭磊,赵玉岩,张世亮,等.骨骼畸形大鼠软骨细胞内胰岛素样生长因子一2的表达研究[J].中国实验动物学报,2008,16(2)：84-87.
[22]王继芳,周凡,曹荣峰.幼龄小鼠骨骼发育的形态学观察[J].上海畜牧兽医通讯,2008,1：26-27.
[23]王文龙,刘阳,李少英等.脱氧雪腐镰刀菌烯醇与人类健康[J].食品研究与开发,2008,29(6)：153-157.
[1]Gyqi SP,Rist B,Gerber SA,et al.Quantitative analysis of complex protein mixtures using isotope-coded affinit tags[J].Nat Biotechnol,1999,17(10):994-999.
[2]Currie I,Davison M. Middleton B.et al.Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology[J]. Proteomics,2001,1(3):377-96.
[3]Husi H,Ward MA,Choudhary JS.et al.Proteomic analysis of NMDA receptor adhesion protein signaling complexes [J].Nat Neurosci,2000,3(7):661-669.
[4]Link AJ,Eng J,Schieltz DM,et al.Direct analysis of protein complexes using mass spectrometry[J].NatBiotecnol,1999,17(7):676-682.
[5]Figeys D,Pinto D.Proteomics on a chip:promising developments[J]. Electr ophoresis,2001,22(2):208-216.
[6]冯海燕,景志忠,房永祥等.双向凝胶电泳技术及其应用[J].生物技术通报,2009,1：59-68.
[7]刘建仁,樊粤光,王海彬等.大鼠骨组织蛋白质组样品提取方法的建立[J].厦门大学学报,2005,44(3)：420-424.
[8]Yueguang Fan, Jianren Liua, Sanying Wang, et al. Functional proteome of bones in rats with osteoporosis following ovariectomy[J]. Life Sciences,2005,76:2893-2901.
[9]Roberta Pastorelli, Donatella Carpi, Luisa Airoldi, et al. Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone[J]. Proteomics,2005, 5:4936-4945.
[10]Melissa A, Schreiweis, Jon P. Butler, et al. A Proteomic Analysis of Adult Rat Bone Reveals the Presence of Cartilage/Chondrocyte Markers[J]. Journal of Cellular Biochemistry,2007,101:466-476.
[11]Xiaogang Jiang, Mingliang Ye, Xinning Jiang, et al. Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis[J]. Journal of Proteome Research,2007,6:2287-2294.
[12]FuYC,Marchal S,Marchal T,et al.Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66kDa protein of Echinococcus granulosusVet Immunol Immun opathol,2000, 74:195-208.
[13]Missich R,Sgorbati B,Donald JL,et al.Transfection of Bifidobacterum longum with pRM2,a constructed Escherichia coli-Bifidobacterum longum shuttle vector [J].Plasmid,1994,32(2):208-11.
[14]王丽华,石玉霞,王文革.T-2毒素致大鼠关节软骨损伤的分子机制研究(Ⅰ)—血清细胞因子检测及其意义[J].中国地方病防治杂志,2009,24(3)：164-166.
[15]杨占田,郭雄,陈静宏等.T-2毒素对软骨细胞NO合成、iNOS表达的影响[J].陕西医学杂志,，200837(9)：1115-1117.
[16]Sutton S, Clutterbuck A, Harris P, et al. The contribution of the synovium, synovial derived inflammatory cytokines and neuropep tides to the pathogenesis of osteoarthritis [J].Vet J,2009,179 (1):10-24.
[17]庞炜,王丽娟,王治伦等.T-2毒素对大鼠骨矿化影响的实验研究[J].中国地方病预防杂志,2000,15(5)：263-265.
[18]Rui Liu, Zhenjun Li, Shujun Bai,et al.Mechanism of Cancer Cell Adaptation to Metabolic Stress[J]. Molecular & Cellular Proteomics,2009,8(1):70-85.
[1]庞炜,王治伦,吕社民,等.真菌毒素DON研究进展[J].中国地方病防治杂志,1996,11(6)：349-351.
[2]霍兴华,赵宝玉,万学攀,等.脱氧雪腐镰刀菌烯醇的毒性研究进展[J].毒理学杂志,2008,22(2)：151-154.
[3]Harvey RB, Kubena LF, Huff WE, et al. Hematologic and immunologic toxicity of deoxynivalenol (DON)-contaminated diets to growing chickens [J]. Bull Environ Contain Toxicol,1991,46(3):410-416. PMID:2032007
[4]Tiemann U, Viergutz T, Jonas L, et al. Influence of the mycotoxins alpha2 and beta2 zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells [J]. Reprod Toxicol,2003,17(2):209-218. PMID: 12642154
[5]Malekinejad H, Schoevers EJ, Daemen IJ, et al. Exposure of oocytes to the fusarium toxins zearalenone and deoxynivalenol causes aneuploidy and abnormal embryo development in pigs [J]. Biol Reprod,2007,77(5): 840-847. PMID:17652666
[6]彭双清,遇秀玲,汪宝珍,等.脱氧雪腐镰刀菌烯醇(DON)致鸡胚软骨细胞损伤及硒的保护效应[J].中国地方病学杂志,1994,13(2)：80-82.
[7]曹峻岭,熊咏民,张矢远,等.真菌毒素DON.T-2和NIV对培养软骨细胞作用的实验研究[J].中国地方病防治杂志,1995,1 0(2)：69.
[8]曹峻岭,熊咏民,张矢远,等.六种真菌毒素对培养软骨细胞的作用[J].医学研究通讯,2001,30(1)：22.
[9]Yue Y, Cao JL, Li Sy, et al. The effect on proteogly can metabolism of deoxynivalenol and selenium in the cultured human fetal chondrocytes in vitro [J]. Academic Journal of Xi'an Jiaotong University,2005,17(2): 151-154.
[10]林林,孙树秋.脱氧雪腐镰刀菌烯醇致Vero细胞核基因组DNA损伤与修复的彗星实验观察[J].中国地方病防治杂志,2004,19(3)：139-141.
[11]刘秀芳,张爱华,张洪英,等.脱氧雪腐镰刀菌烯醇诱导血管内皮细胞凋亡及其凋亡相关因子的初步研究[J].畜牧兽医学报,2008,39(6)：778-783.
[12]Calvert TW, Aidoo KE, Candlish AGG, et al. Comparison of in vitro cytotoxicity of Fusarium mycotoxins, deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines [J]. Mycopathologia, 2005,159(3):413-419. PMID:15883728
[13]李群伟,侯海峰,李亚鲁,等.脱氧雪腐镰刀菌烯醇致家兔多器官损伤的实验观察[J].中国地方病防治杂志,2007,22(1)：19-22.
[14]Tiemanna U, Dannenberger D, Jonas L, et al. The effect of feeding a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZON) on the spleen and liver of sow and fetus from day 35 to 70 of gestation [J]. Toxicol Lett,2008,179(3):113-117. PMID:18550300
[15]彭双清,杨进生.镰刀菌毒素脱氧雪腐镰刀菌烯醇对心肌细胞钙通道的阻滞作用[J].中国预防医学杂志,2004,5(4)：24 1-243.
[16]邢欣,刘静,邢凌霄等.脱氧雪腐镰刀菌烯醇对胃癌细胞增殖及细胞周期的影响[J].细胞生物学杂志,2007,29(5)：729-735.
[17]刘静,邢欣,邢凌霄等.DON对胃癌细胞H GC227细胞周期和凋亡的影响[J].肿瘤防治研究,2007,34(12)：897-900.
[18]王加生,徐达道.赤霉病麦中毒研究Ⅳ.赤霉病麦粗毒素的致畸与致突变研究[J].1986,5(1)：52-62.
[19]Vesely D, Vesela D. Embryo toxic effects of a combination zearalenone and vomitoxin (4-dioxynivalenone) on the chick embryo [J]. Vet Med Phaha, 1995,40(9):279-281. PMID:8659095
[20]Walser MM, Morris VC, Levander OA,et al. Effect of dietary selenium on the development of Fusarium-induced tibial dyschondroplasia in broiler chickens[J]. Avian Disases,1988,32:84-88.PMID:3382383
[21]Morrissey RE, Norred WP, Vesonder RF. Subchronic toxicity of vomitoxin in Sprague-Dawley rats [J]. Food Chem Toxicol,1985,23(11):995-999. PMID:2934304
[22]Debouck C, Haubruge E, Bollaerts P, et al. Skeletal deformities induced by the intraperitoneal administration of deoxynivalenol (vomitoxin) in mice [J]. Intl Ortho,2001,25(3):194-198. PMID:11482540
[23]Collins TF, Sprando RL, Black TN, et al. Effects of deoxynivalenol (DON, vomitoxin) on in utero development in rats [J]. Food Chem Toxicol,2006, 44(6):747-757. PMID:16325976
[24]Khera KS, Whalen C, Angers G, et al. Embryotoxicity of 4-deoxynivalenol (vomitoxin) in mice [J]. Bull Environm Contain Toxicol,1982,29 (4): 487-491. PMID:7171859
[25]王治伦.大骨节病的4种病因学说同步研究[J].西安交通大学学报(医学版),2005,26(1)：1-7.
[26]张帆,吴志远,吴健丽,等.食管癌高发区粮食中镰刀菌毒素的含量及其致突变作用[J].中国预防医学杂志,2000,34(1)：53.
[27]李东华,孙艺,罗雪云,等.脱氧雪腐镰刀菌烯醇(DON)对中国仓鼠肺纤维CHL细胞的遗传毒性[J].中国食品卫生杂志,1992,4(2)：40-43.
[28]许兰文,抑启沛,邵玉芬,等.脱氧雪腐镰刀菌烯醇(CBD1)的毒性探讨：CBD1对DNA合成的抑制作用观察[J].中国食品卫生杂志,1991,3(2)：8-10.
[29]郭红卫,许洁.镰刀菌毒素与黄曲霉毒素的联合毒性研究[J].中国食品卫生杂志,1997,9(1)：10-12.
[30]李斌,郭红卫.镰刀菌毒素与黄曲霉毒素的联合毒性研究—AFB1和DON的RDS实验[J].卫生研究,2000,29(6)：393-395.
[31]黄向华,张祥宏,李月红,等.杂色曲霉素和脱氧雪腐镰刀菌烯醇对小鼠致癌作用的研究[J].中华肿瘤杂志,2004,26(12)：705-708.
[32]Iverson F, Armstrong C, Nera E, et al. Chronic feeding study of deoxynivalenol in B6C3F1 male and female mice[J].Theratog Carcinog Mutagen,1995,15(6):283-306. PMID:8732880
[33]Lambert LA, Hines FA, Eppley RM. Lack of initiation and promotion potential of deoxynivalenol for skin tumorigenesis in Sencar mice [J].Food chem Toxical,1995,33(3):217-222. PMID:7896232
[2]Pestka JJ.Smolinski AT.Deoxynivalenol:toxicology and potential effects on humans [J].J.Toxicol.Enyiron.Health B Crit.Rev,2005,8(1):39-69.
[3]Centin Y,Bullerman LB.Cytotoxicity of Fusarium mycotoxins to mammalian cell cultures as determined by the MTT bioassay[J].Food Chem Toxicol,2005,43:754-764.
[4]Bimezok D.Doll S.Rau H, et al.The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cell in vivo and in vitro [J].Immunobiology,2007,212:655-666.
[5]Peska JJ,Amuzie CJ.Tissue distribution and proinflammatory cytokine gene expression following acute oral exposure to deoxynivalenol:Comparison of weanling and adult mice[J].Food and Chemical Toxicology,2008,46:2826-2831.
[6]Doll S,Schrickxa JA,Danickeb S, et al.Deoxynivalenol-induced cytotoxicity, cytokins and related genes in unstimulated or lipopolysaccharide stimula-ted primary porcine macrophages[J].Toxicology Letter,2009,284:97-106.
[7]李群伟,侯海峰,李亚鲁,等.脱氧雪腐镰刀菌烯醇致家兔多器官损伤的实验观察[J].中国地方病防治杂志,2007,22(1)：19-22.
[8]Konigs M,Lencxyk M,Schwerdt G, et al.Cytotoxicity,metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture[J].Toxicology,2007,240:48-59.
[9]Yang H,Chung D H,KimY B, et al.Ribotoxic mycotoxin deoxynivalenol induces G2/M cell cycle arrest via p21Cip/WAFI mRNA stabilization in human epithelial cells[J].Toxicology,2009,243:145-154.
[10]Bony S,Carcelen M,OlivierL, et al.Genotoxicity assessment of deoxynival-enol in the Caco-2 cell line model using the Comet assay[J].Toxicology Letter,2006,166:67-76.
[11]Manda G,Neagu M,Constantin C, et al.Immunotoxicology of mycotoxins produced by Fusarium fungi-Low concentrations of deoxynivalenol interfere with nucleotide metabolism[J].Toxicology Letter,2007,172:S49.
[12]王加生,徐达道.赤霉病麦中毒研究ⅣV.赤霉病麦粗毒素的致畸与致突变研究[J].真菌学报,1986,5(1)：52-62.
[13]庞炜,王志伦,吕社民,等.真菌毒素DON研究进展[J].中国地方病防治杂志,1996,11 (6)：349-351.
[14]Vesely D, Vesela D. Embryo toxic effects of a combination zear-alenone and vomitoxin(4-dioxynivalenone) on the chick embryl [J].Vet Med Phaha,1995, 40(9):279-281.
[15]Morrissey RE.Norred WP.Vesonder RF.Subchronic toxicity of vomitoxin in Sprague-Dawley rats[J].Food Chem Toxicol,1985,23(11):995-999.
[16]Debouck.C.,Haubruge.E, Bollaerts.P, et al.Skeletal deformities induced by the intrapertoneal administration of deoxynivalenol (vomitoxin) in mice [J].International Orthopaedics,2001,25(3):194-198.
[17]Collins TF,Sprando RL.Black TN, et al. Effects of deoxynivalenol (DON, vomitoxin) on in utero development in rats[J].Food Chem Toxicol,2006,44(6):747-757.
[18]Khera KS, Whalen C, Angers G, et al. Embryotoxicity of 4-Deoxynivalenol (Vomitoxin) in Mice[J].Bull Environm Contain Toxicol.1982,29 (4):487-491.
[19]李勇,王维林,袁正伟,等.一种简捷可靠的胎鼠骨和软骨双重染色技术[J].中国医科大学学报,2004,33(2)：189-190.
[20]Young AD, Phipps DE, Astroff AB. Large2scale double2staining of rat fetal skeletons using Alizarin Red S and Alcian Blue [J].Teratology,2000,61 (4):273-276.
[21]郭磊,赵玉岩,张世亮,等.骨骼畸形大鼠软骨细胞内胰岛素样生长因子一2的表达研究[J].中国实验动物学报,2008,16(2)：84-87.
[22]王继芳,周凡,曹荣峰.幼龄小鼠骨骼发育的形态学观察[J].上海畜牧兽医通讯,2008,1：26-27.
[23]王文龙,刘阳,李少英等.脱氧雪腐镰刀菌烯醇与人类健康[J].食品研究与开发,2008,29(6)：153-157.
[1]Gyqi SP,Rist B,Gerber SA,et al.Quantitative analysis of complex protein mixtures using isotope-coded affinit tags[J].Nat Biotechnol,1999,17(10):994-999.
[2]Currie I,Davison M. Middleton B.et al.Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology[J]. Proteomics,2001,1(3):377-96.
[3]Husi H,Ward MA,Choudhary JS.et al.Proteomic analysis of NMDA receptor adhesion protein signaling complexes [J].Nat Neurosci,2000,3(7):661-669.
[4]Link AJ,Eng J,Schieltz DM,et al.Direct analysis of protein complexes using mass spectrometry[J].NatBiotecnol,1999,17(7):676-682.
[5]Figeys D,Pinto D.Proteomics on a chip:promising developments[J]. Electr ophoresis,2001,22(2):208-216.
[6]冯海燕,景志忠,房永祥等.双向凝胶电泳技术及其应用[J].生物技术通报,2009,1：59-68.
[7]刘建仁,樊粤光,王海彬等.大鼠骨组织蛋白质组样品提取方法的建立[J].厦门大学学报,2005,44(3)：420-424.
[8]Yueguang Fan, Jianren Liua, Sanying Wang, et al. Functional proteome of bones in rats with osteoporosis following ovariectomy[J]. Life Sciences,2005,76:2893-2901.
[9]Roberta Pastorelli, Donatella Carpi, Luisa Airoldi, et al. Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone[J]. Proteomics,2005, 5:4936-4945.
[10]Melissa A, Schreiweis, Jon P. Butler, et al. A Proteomic Analysis of Adult Rat Bone Reveals the Presence of Cartilage/Chondrocyte Markers[J]. Journal of Cellular Biochemistry,2007,101:466-476.
[11]Xiaogang Jiang, Mingliang Ye, Xinning Jiang, et al. Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis[J]. Journal of Proteome Research,2007,6:2287-2294.
[12]FuYC,Marchal S,Marchal T,et al.Cellular immune response of lymph nodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66kDa protein of Echinococcus granulosusVet Immunol Immun opathol,2000, 74:195-208.
[13]Missich R,Sgorbati B,Donald JL,et al.Transfection of Bifidobacterum longum with pRM2,a constructed Escherichia coli-Bifidobacterum longum shuttle vector [J].Plasmid,1994,32(2):208-11.
[14]王丽华,石玉霞,王文革.T-2毒素致大鼠关节软骨损伤的分子机制研究(Ⅰ)—血清细胞因子检测及其意义[J].中国地方病防治杂志,2009,24(3)：164-166.
[15]杨占田,郭雄,陈静宏等.T-2毒素对软骨细胞NO合成、iNOS表达的影响[J].陕西医学杂志,，200837(9)：1115-1117.
[16]Sutton S, Clutterbuck A, Harris P, et al. The contribution of the synovium, synovial derived inflammatory cytokines and neuropep tides to the pathogenesis of osteoarthritis [J].Vet J,2009,179 (1):10-24.
[17]庞炜,王丽娟,王治伦等.T-2毒素对大鼠骨矿化影响的实验研究[J].中国地方病预防杂志,2000,15(5)：263-265.
[18]Rui Liu, Zhenjun Li, Shujun Bai,et al.Mechanism of Cancer Cell Adaptation to Metabolic Stress[J]. Molecular & Cellular Proteomics,2009,8(1):70-85.
[1]庞炜,王治伦,吕社民,等.真菌毒素DON研究进展[J].中国地方病防治杂志,1996,11(6)：349-351.
[2]霍兴华,赵宝玉,万学攀,等.脱氧雪腐镰刀菌烯醇的毒性研究进展[J].毒理学杂志,2008,22(2)：151-154.
[3]Harvey RB, Kubena LF, Huff WE, et al. Hematologic and immunologic toxicity of deoxynivalenol (DON)-contaminated diets to growing chickens [J]. Bull Environ Contain Toxicol,1991,46(3):410-416. PMID:2032007
[4]Tiemann U, Viergutz T, Jonas L, et al. Influence of the mycotoxins alpha2 and beta2 zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells [J]. Reprod Toxicol,2003,17(2):209-218. PMID: 12642154
[5]Malekinejad H, Schoevers EJ, Daemen IJ, et al. Exposure of oocytes to the fusarium toxins zearalenone and deoxynivalenol causes aneuploidy and abnormal embryo development in pigs [J]. Biol Reprod,2007,77(5): 840-847. PMID:17652666
[6]彭双清,遇秀玲,汪宝珍,等.脱氧雪腐镰刀菌烯醇(DON)致鸡胚软骨细胞损伤及硒的保护效应[J].中国地方病学杂志,1994,13(2)：80-82.
[7]曹峻岭,熊咏民,张矢远,等.真菌毒素DON.T-2和NIV对培养软骨细胞作用的实验研究[J].中国地方病防治杂志,1995,1 0(2)：69.
[8]曹峻岭,熊咏民,张矢远,等.六种真菌毒素对培养软骨细胞的作用[J].医学研究通讯,2001,30(1)：22.
[9]Yue Y, Cao JL, Li Sy, et al. The effect on proteogly can metabolism of deoxynivalenol and selenium in the cultured human fetal chondrocytes in vitro [J]. Academic Journal of Xi'an Jiaotong University,2005,17(2): 151-154.
[10]林林,孙树秋.脱氧雪腐镰刀菌烯醇致Vero细胞核基因组DNA损伤与修复的彗星实验观察[J].中国地方病防治杂志,2004,19(3)：139-141.
[11]刘秀芳,张爱华,张洪英,等.脱氧雪腐镰刀菌烯醇诱导血管内皮细胞凋亡及其凋亡相关因子的初步研究[J].畜牧兽医学报,2008,39(6)：778-783.
[12]Calvert TW, Aidoo KE, Candlish AGG, et al. Comparison of in vitro cytotoxicity of Fusarium mycotoxins, deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines [J]. Mycopathologia, 2005,159(3):413-419. PMID:15883728
[13]李群伟,侯海峰,李亚鲁,等.脱氧雪腐镰刀菌烯醇致家兔多器官损伤的实验观察[J].中国地方病防治杂志,2007,22(1)：19-22.
[14]Tiemanna U, Dannenberger D, Jonas L, et al. The effect of feeding a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZON) on the spleen and liver of sow and fetus from day 35 to 70 of gestation [J]. Toxicol Lett,2008,179(3):113-117. PMID:18550300
[15]彭双清,杨进生.镰刀菌毒素脱氧雪腐镰刀菌烯醇对心肌细胞钙通道的阻滞作用[J].中国预防医学杂志,2004,5(4)：24 1-243.
[16]邢欣,刘静,邢凌霄等.脱氧雪腐镰刀菌烯醇对胃癌细胞增殖及细胞周期的影响[J].细胞生物学杂志,2007,29(5)：729-735.
[17]刘静,邢欣,邢凌霄等.DON对胃癌细胞H GC227细胞周期和凋亡的影响[J].肿瘤防治研究,2007,34(12)：897-900.
[18]王加生,徐达道.赤霉病麦中毒研究Ⅳ.赤霉病麦粗毒素的致畸与致突变研究[J].1986,5(1)：52-62.
[19]Vesely D, Vesela D. Embryo toxic effects of a combination zearalenone and vomitoxin (4-dioxynivalenone) on the chick embryo [J]. Vet Med Phaha, 1995,40(9):279-281. PMID:8659095
[20]Walser MM, Morris VC, Levander OA,et al. Effect of dietary selenium on the development of Fusarium-induced tibial dyschondroplasia in broiler chickens[J]. Avian Disases,1988,32:84-88.PMID:3382383
[21]Morrissey RE, Norred WP, Vesonder RF. Subchronic toxicity of vomitoxin in Sprague-Dawley rats [J]. Food Chem Toxicol,1985,23(11):995-999. PMID:2934304
[22]Debouck C, Haubruge E, Bollaerts P, et al. Skeletal deformities induced by the intraperitoneal administration of deoxynivalenol (vomitoxin) in mice [J]. Intl Ortho,2001,25(3):194-198. PMID:11482540
[23]Collins TF, Sprando RL, Black TN, et al. Effects of deoxynivalenol (DON, vomitoxin) on in utero development in rats [J]. Food Chem Toxicol,2006, 44(6):747-757. PMID:16325976
[24]Khera KS, Whalen C, Angers G, et al. Embryotoxicity of 4-deoxynivalenol (vomitoxin) in mice [J]. Bull Environm Contain Toxicol,1982,29 (4): 487-491. PMID:7171859
[25]王治伦.大骨节病的4种病因学说同步研究[J].西安交通大学学报(医学版),2005,26(1)：1-7.
[26]张帆,吴志远,吴健丽,等.食管癌高发区粮食中镰刀菌毒素的含量及其致突变作用[J].中国预防医学杂志,2000,34(1)：53.
[27]李东华,孙艺,罗雪云,等.脱氧雪腐镰刀菌烯醇(DON)对中国仓鼠肺纤维CHL细胞的遗传毒性[J].中国食品卫生杂志,1992,4(2)：40-43.
[28]许兰文,抑启沛,邵玉芬,等.脱氧雪腐镰刀菌烯醇(CBD1)的毒性探讨：CBD1对DNA合成的抑制作用观察[J].中国食品卫生杂志,1991,3(2)：8-10.
[29]郭红卫,许洁.镰刀菌毒素与黄曲霉毒素的联合毒性研究[J].中国食品卫生杂志,1997,9(1)：10-12.
[30]李斌,郭红卫.镰刀菌毒素与黄曲霉毒素的联合毒性研究—AFB1和DON的RDS实验[J].卫生研究,2000,29(6)：393-395.
[31]黄向华,张祥宏,李月红,等.杂色曲霉素和脱氧雪腐镰刀菌烯醇对小鼠致癌作用的研究[J].中华肿瘤杂志,2004,26(12)：705-708.
[32]Iverson F, Armstrong C, Nera E, et al. Chronic feeding study of deoxynivalenol in B6C3F1 male and female mice[J].Theratog Carcinog Mutagen,1995,15(6):283-306. PMID:8732880
[33]Lambert LA, Hines FA, Eppley RM. Lack of initiation and promotion potential of deoxynivalenol for skin tumorigenesis in Sencar mice [J].Food chem Toxical,1995,33(3):217-222. PMID:7896232