兔上肢骨折愈合的蛋白质组学双向电泳分析
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摘要
目的:利用双向电泳技术,获得兔骨折愈合不同时期(2w,4w,8w,12w)的骨痂蛋白和正常骨组织的蛋白质双向电泳图谱,动态的观察骨痂组织中蛋白质含量、种类与正常骨组织的差异,找出表达差异的蛋白质点,为后续进行质谱分析,鉴定出参与骨折愈合过程的特异性蛋白质奠定基础。
方法:选择新西兰大白兔40只,体重2-2.2kg,随机分成4组,每组10只。建立兔桡骨的骨折模型,分别于2w、4w、8w、12w处死动物,截取骨痂组织及对侧正常骨组织。使用蛋白质裂解液,以正常骨组织为对照组,2w、4w、8w、12w的骨痂蛋白为实验组,分别提取各组骨组织的总蛋白,检测各组蛋白提取液的浓度,用SDS-PAGE凝胶电泳初步观察,双向凝胶电泳分离蛋白质和考马斯亮蓝染色后扫描获取高清晰的蛋白质图谱,再经计算机软件对比分析,得到表达差异的蛋白质点。
结果:各实验组和对照组的双向凝胶电泳图谱分辨率高,重复性好。骨折愈合不同时期获得的蛋白质点个数:2周(843±29),4周(727±36),8周(785±30),12周(737±42),对照组蛋白点数为(710±25)。以对照组的双向电泳图谱为比较对象,实验组匹配蛋白点相对含量的比值大于2或者小于0.5则认为是差异蛋白质点,从而得出蛋白质差异点数目是: 22(2周),26(4周),24(8周),21(12周)。
结论:本研究建立了分辨率高,且重复性好的兔骨折愈合蛋白质组学双向凝胶电泳图谱,发现了许多在骨折愈合过程中与对照组相比表达差异的蛋白质。为进一步分析鉴定骨折愈合相关的特异蛋白,探寻骨折愈合的机制奠定了基础。
OBJECTIVE Using two-dimensional electrophoresis technology to obtain Two-dimensional electrophoresis map of proteins which callus protein in different stages (2w,4w,8w,12w)of fracture healing and normal bone protein . Observating dynamicly the differences of callus tissue and normal bone in protein species and protein contents, finding differentially expressed proteins. For the subsequent mass spectrometry analysis of proteins,identified specific proteins in fracture healing.
METHODS Select 40 New Zealand white rabbits about 2-2.2kg, were divided into 4 groups randomly, which 10 in each group. Establish the fracture model of the rabbit’s radial bone, and kill all the animals in 2w, 4w, 8w, 12w later animals respectively . the interception of callus tissue and contralateral. Collected the radius callus tissue and the contralateral normal bone tissue as a control group. Lysis buffer used to extract total protein of bone tissues in each experimental group and the control group. Detected the concentration of protein in each group. Proteins were separated by SDS-PAGE gel electrophoresis and two-dimensional gel electrophoresis。Then it was stained with Coomassie brilliant blue to obtain high-resolution protein mapping。Scanned images by computer software, finally found that the protein differences in the experimental group and control group。
RESULTS Two-dimensional gel electrophoresis profiles of the experimental groups and control groups hava high resolution and reproducibility。Protein spots are different in different periods of fracture healing:2w(843±29),4w(727±36),8w(785±30),12w(737±42)。Protein spots in the control group:(710±25)。To the control group as the reference object, The matching protein points of the experimental group have the content of the protein more than 2 or less than 0.5 which it is different proteins。The quantity of the differents protein in all experimental groups is:22(2W),26(4W),24(8W),21(12W)。
CONCLUSIONS This experiment established a high-resolution and repeatability of two-dimensional electrophoresis map in the rabbit’s fracture healing. Found a number of different proteins associated with the fracture healing. IT is important to further analyzed and identified specific proteins related to fracture healing,and it is useful to clarify the mechanisms of fracture healing of the foundation
方法:选择新西兰大白兔40只,体重2-2.2kg,随机分成4组,每组10只。建立兔桡骨的骨折模型,分别于2w、4w、8w、12w处死动物,截取骨痂组织及对侧正常骨组织。使用蛋白质裂解液,以正常骨组织为对照组,2w、4w、8w、12w的骨痂蛋白为实验组,分别提取各组骨组织的总蛋白,检测各组蛋白提取液的浓度,用SDS-PAGE凝胶电泳初步观察,双向凝胶电泳分离蛋白质和考马斯亮蓝染色后扫描获取高清晰的蛋白质图谱,再经计算机软件对比分析,得到表达差异的蛋白质点。
结果:各实验组和对照组的双向凝胶电泳图谱分辨率高,重复性好。骨折愈合不同时期获得的蛋白质点个数:2周(843±29),4周(727±36),8周(785±30),12周(737±42),对照组蛋白点数为(710±25)。以对照组的双向电泳图谱为比较对象,实验组匹配蛋白点相对含量的比值大于2或者小于0.5则认为是差异蛋白质点,从而得出蛋白质差异点数目是: 22(2周),26(4周),24(8周),21(12周)。
结论:本研究建立了分辨率高,且重复性好的兔骨折愈合蛋白质组学双向凝胶电泳图谱,发现了许多在骨折愈合过程中与对照组相比表达差异的蛋白质。为进一步分析鉴定骨折愈合相关的特异蛋白,探寻骨折愈合的机制奠定了基础。
OBJECTIVE Using two-dimensional electrophoresis technology to obtain Two-dimensional electrophoresis map of proteins which callus protein in different stages (2w,4w,8w,12w)of fracture healing and normal bone protein . Observating dynamicly the differences of callus tissue and normal bone in protein species and protein contents, finding differentially expressed proteins. For the subsequent mass spectrometry analysis of proteins,identified specific proteins in fracture healing.
METHODS Select 40 New Zealand white rabbits about 2-2.2kg, were divided into 4 groups randomly, which 10 in each group. Establish the fracture model of the rabbit’s radial bone, and kill all the animals in 2w, 4w, 8w, 12w later animals respectively . the interception of callus tissue and contralateral. Collected the radius callus tissue and the contralateral normal bone tissue as a control group. Lysis buffer used to extract total protein of bone tissues in each experimental group and the control group. Detected the concentration of protein in each group. Proteins were separated by SDS-PAGE gel electrophoresis and two-dimensional gel electrophoresis。Then it was stained with Coomassie brilliant blue to obtain high-resolution protein mapping。Scanned images by computer software, finally found that the protein differences in the experimental group and control group。
RESULTS Two-dimensional gel electrophoresis profiles of the experimental groups and control groups hava high resolution and reproducibility。Protein spots are different in different periods of fracture healing:2w(843±29),4w(727±36),8w(785±30),12w(737±42)。Protein spots in the control group:(710±25)。To the control group as the reference object, The matching protein points of the experimental group have the content of the protein more than 2 or less than 0.5 which it is different proteins。The quantity of the differents protein in all experimental groups is:22(2W),26(4W),24(8W),21(12W)。
CONCLUSIONS This experiment established a high-resolution and repeatability of two-dimensional electrophoresis map in the rabbit’s fracture healing. Found a number of different proteins associated with the fracture healing. IT is important to further analyzed and identified specific proteins related to fracture healing,and it is useful to clarify the mechanisms of fracture healing of the foundation
引文
[1]王莹,王全军,廖明阳.蛋白质组学技术在药物肝毒性研究中的应用[J]。中国新药杂志,2008,17(4):348-352
[2] Yu-Chang Tyan, Hsin-YiWu, Wu-WeiLai, a1.Proteomic Profiling of Human Pleural Effusion Using Two-Dimensional Nano Liquid Chromatography Tandem Mass Spectrometry.[J]. Journal of Proteome Research ,2005, 4,1274-1286
[3] Yuan Li, Da-Fang Wan, Jian-Jia Su,a1.Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews. [J] WORLD JOURNAL OF GASTROENTEROLOGY,2004;10(4):497—504
[4]景爱红。双向电泳技术在蛋白质组研究中的应用体会[J]。解剖学杂志,2 0 0 8,3 1(5):728-729
[5]高堂成,康庆林,张春才。上肢骨干骨折内固定实验动物模型的建立[J]。中国骨伤,2004,17(5):267-269
[6]罗小云,吴庆华。不同样品制备方法对血浆双向凝胶电泳图谱的影响[J]。中华实验外科杂志,2007,24(2):140-141
[7]师杰,卢朝辉,崔全才。甲状腺乳头状癌的蛋白质组学研究[J]。中华病理学杂志,2008,37(7):465-470
[8]宁宁,刘巨超,夏季等。肝大部切除术后小鼠血浆蛋白质组双向凝胶电泳图谱差异分析[J]。中华实验外科杂志,2006,23(4):557-559
[9]刘秋均,李洪,严冬梅等。小鼠肝癌细胞H22膜蛋白组的双向电泳[J]。中华肝脏病杂志,2009,17(4):280-283
[10]贾宇峰刘少君郭尧君。双向电泳图像分析软件[J]。现代科学仪器,2000,5:20-23
[11]靳胜,张曼,张秀敏等。以双向电泳为基础的肺癌组织差异蛋白分析策略[J]。第三军医大学学报,2009,31(7):644-645
[12]林坚平骨折愈合及早期诊断的研究进展[J]。中国矫形外科杂志,2009,17(24):1876-1878
[13]刘杭,谢嵘,姚鏊等。中枢神经系统血管母细胞瘤细胞与人脑神经元细胞差异蛋白质分析[J]。分析化学研究报告,2009,37(10):1439-1444
[14]刘国红,杨继要,呼琳等。人胃贲门腺癌组织和正常胃黏膜组织蛋白质组双向电泳图谱的差异分析[J]。第三军医大学学报,2009,31(17):1712-1713
[15]张虹,牛屹东,冯捷。应用双向电泳、免疫印迹和质谱技术筛选子宫内膜异位症标志物[J]。北京大学学报(医学版),2005,34(4):366-370
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[18]康献刚,王志强。骨折愈合过程中细胞因子的作用[J]。中国修复重建外科杂志,2008,22(7):877-879
[19]卢卫忠唐康来赵麟丰等。外源性TGF—13和BMP对兔尺骨骨折愈合作用的研究[J]。中国矫形外科杂志,2005,5,(10):770-772
[20]陈昱,陈宗雄,徐皓等。骨折愈合机制的现代研究[J]。中国组织工程研究与临床康复,2008,12(50):9951-9956
[21]周颖,侯树勋,陈秉等。骨髓问充质干细胞定向诱导成骨分化的蛋白质组学分析[J]。中国骨肿瘤骨病杂志,2009,8(5):296-299
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[6] Domon B,Aebersold R.Mass spectrometry and protein analysis[J].Science,2006,312(5771):212.
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[3] Yuan Li, Da-Fang Wan, Jian-Jia Su,a1.Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews. [J] WORLD JOURNAL OF GASTROENTEROLOGY,2004;10(4):497—504
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[6]罗小云,吴庆华。不同样品制备方法对血浆双向凝胶电泳图谱的影响[J]。中华实验外科杂志,2007,24(2):140-141
[7]师杰,卢朝辉,崔全才。甲状腺乳头状癌的蛋白质组学研究[J]。中华病理学杂志,2008,37(7):465-470
[8]宁宁,刘巨超,夏季等。肝大部切除术后小鼠血浆蛋白质组双向凝胶电泳图谱差异分析[J]。中华实验外科杂志,2006,23(4):557-559
[9]刘秋均,李洪,严冬梅等。小鼠肝癌细胞H22膜蛋白组的双向电泳[J]。中华肝脏病杂志,2009,17(4):280-283
[10]贾宇峰刘少君郭尧君。双向电泳图像分析软件[J]。现代科学仪器,2000,5:20-23
[11]靳胜,张曼,张秀敏等。以双向电泳为基础的肺癌组织差异蛋白分析策略[J]。第三军医大学学报,2009,31(7):644-645
[12]林坚平骨折愈合及早期诊断的研究进展[J]。中国矫形外科杂志,2009,17(24):1876-1878
[13]刘杭,谢嵘,姚鏊等。中枢神经系统血管母细胞瘤细胞与人脑神经元细胞差异蛋白质分析[J]。分析化学研究报告,2009,37(10):1439-1444
[14]刘国红,杨继要,呼琳等。人胃贲门腺癌组织和正常胃黏膜组织蛋白质组双向电泳图谱的差异分析[J]。第三军医大学学报,2009,31(17):1712-1713
[15]张虹,牛屹东,冯捷。应用双向电泳、免疫印迹和质谱技术筛选子宫内膜异位症标志物[J]。北京大学学报(医学版),2005,34(4):366-370
[16]张晓娜监测骨折愈合的生物化学骨标志物[J]。中国全科医学,2006,9(1):65-67
[17]张谚强,张松林。内皮细胞生长因子及碱性成纤维细胞生长因子对兔桡骨骨折愈合影响的比较[J]。中国组织工程研究与临床康,2008,33(12):6453-6456
[18]康献刚,王志强。骨折愈合过程中细胞因子的作用[J]。中国修复重建外科杂志,2008,22(7):877-879
[19]卢卫忠唐康来赵麟丰等。外源性TGF—13和BMP对兔尺骨骨折愈合作用的研究[J]。中国矫形外科杂志,2005,5,(10):770-772
[20]陈昱,陈宗雄,徐皓等。骨折愈合机制的现代研究[J]。中国组织工程研究与临床康复,2008,12(50):9951-9956
[21]周颖,侯树勋,陈秉等。骨髓问充质干细胞定向诱导成骨分化的蛋白质组学分析[J]。中国骨肿瘤骨病杂志,2009,8(5):296-299
[1] Aggarwal K,lee KH.Functional genomics and proteomics as a foundation for systems biology.Briefings in Functional Genomics and Proteomics 2003,2(3):175-184
[2] Patterson SD,Aebersold RH.Proteomics:the first decade and beyond[J].Nature Genetics,2003,33:311—323.
[3]王封郭尧军.蛋白质组学及技术发展[J].现代科学仪器2006,5:9-10.
[4]赵海豹林汝仙等.蛋白质组学研究相关技术及进展[J].生物技术通讯,2008,19(6):903-905
[5]张健泓.陈优生.高效液相色谱法概论[J].中国医药导报,2008,8,5(22):28-29
[6] Domon B,Aebersold R.Mass spectrometry and protein analysis[J].Science,2006,312(5771):212.
[7] Junmin Peng, Joshua E, Elias t a1.Evaluation of Multidimensional Chromatography Coupled with Tandem Mass Spectrometry (LC/LC-MS/MS) for Large-Scale Protein Analysis: The Yeast Proteome [J]. Proteome Research 2003, 2, 43-50
[8] Dijkstra M,Vonk R J,Jansen R C.SELDI—TOF mass spectra:a view on sources of variation[J].J Chromatogr B,2007,847(1):12-23.
[9] Revest M ,Donaghy L,Cabillic F,et a1.Comparison of theimmunomodulatory effects of L.donovani and L.major excreted-secreted antigens,particulate and soluble extracts and viable parasites on human dendritic cells[J]. Vaccine,2008,26(48):6119-6123.
[10]高媛.后基因组时代的生物信息学发展[J].中国科技信息,2009,10:225-226
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