脑损伤并骨折大鼠的骨折骨痂的蛋白质组学研究

.Patients of fracture associated with cerebral trauma have commonly been found to have heterotopic ossification and apparently shorter heal time than those with only fracture. Although a lot of researches about this have been done by domestic and foreign scholars, the mechanism has not been known so far. In our preliminary studies, it has been found that many functional proteins in the osteotylus of the fracture associated with cerebral trauma are changing over time obviously. Protein is the direct perpetrator of vital functions, and is also the basis of researches on life. However, at present there is not any dynamic proteomics research about the osteotylus of the fracture associated with cerebral trauma. We will use the two-dimensional gel electrophoresis-matrix assisted with laser desorption/ionization time-of-flying mass spectrometry to create the dynamic different protein expression profiles about the osteotylus of the fracture associated with cerebral trauma in this project. And we will elect the functional proteins which express differentially in different time through the corroboration between the two-dimensional gel electrophoresis and the confirmation about translation and transcription of differential proteins. Then we'll identify the function of important different proteins by using gene transfection or RNA interference. Hence we can discover new regulatory proteins, understand the interacting relationship among various proteins, analyse the role they played during fracture healing, explore the new protein regulatory site and discuss the fracture healing acceleration mechanism of fracture associated with cerebral trauma. This mechanism will help to improve clinical outcomes of fracture and to prevent and treat the osteoporosis and heterotopic ossification, and so on.

临床上常见脑损伤并骨折患者较单纯骨折患者的骨折愈合时间明显缩短, 迄今为止, 其机理尚不明.我们的前期实验研究发现脑损伤并骨折大鼠的骨折骨痂中有多种功能蛋白质随时间而明显变化。蛋白质是生命功能的直接实施者，是研究生命的基础，但目前尚无脑损伤并骨折大鼠的骨折骨痂的动态蛋白质组学研究。本项目拟采用双向凝胶电泳-飞行时间质谱技术创建动态的脑损伤并骨折大鼠和骨折大鼠的骨痂差异蛋白表达谱,通过对差异蛋白分子翻译水平和转录水平的验证与双向凝胶电泳的定量结果相印证, 选出不同时间点有差异表达的功能蛋白,运用基因转染技术或RNA干扰技术，对筛选出的重要差异分子进行蛋白功能验证，发现新的调控蛋白，了解各种蛋白质之间相互作用的关系，分析它们在骨折愈合加速过程中的作用，来探讨脑损伤并骨折病人的骨折愈合加速机制和探索新的蛋白调控位点。利用这种机制有助于提高临床骨折的疗效，防治骨质疏松。

项目背景： 临床常见脑损伤并骨折患者的骨折愈合时间明显缩短, 甚至出现异位骨化现象，国内外学者对此进行了大量研究,但迄今为止,尚不明其机理.我们早期的实验研究发现此种骨折骨痂中有多种功能蛋白质随时间而明显变化。蛋白质是生命功能的直接实施者，是研究生命的基础。但过往尚无脑损伤并骨折的骨折骨痂的动态蛋白质组学研究。.本实验目的：探讨脑损伤并骨折病人的骨折愈合加速机制,利用这种机制有助于提高临床骨折的疗效，防止骨质疏松、骨不连或骨折延期愈合的发生。. 本实验主要研究内容和方法：A 建立大鼠脑损伤模型同时制造大鼠胫骨骨折模型，即建立伴脑损伤的骨折模型。 B 采用双向凝胶电泳-飞行时间质谱技术创建动态的脑损伤并骨折大鼠的骨痂差异蛋白表达谱,对差异蛋白分子进行验证， C 观察模型不同时期骨折骨痂的影像学变化和骨折骨痂中的骨细胞变化情况。 E 在成骨细胞培养中，加入外源性在对照组和实验组中有显著骨再生表达差异的蛋白以及脑损伤动物血清，通过细胞计数的方法了解成骨细胞分化过程中受这些差异蛋白对其的影响情况。.实验结果：通过实验建立的脑外伤合并骨折模型的骨折骨痂的差异蛋白发现，有28种蛋白在各个时间检测阶段明显上调，17种蛋白表达下调，大部分蛋白表达呈现波动表达。结论：28种蛋白达上调和17种蛋白表达下调的基因，可能是促进脑损伤合并骨折大鼠骨折愈合加速的主要基因。利用这种机制有助于提高临床骨折的疗效，防止骨质疏松、骨不连或骨折延期愈合的发生。