【作者】 秦志；

【导师】 苏伟；

【作者基本信息】 广西医科大学 ， 创伤骨科， 2010， 硕士

【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。更多还原

【Abstract】 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更多还原