应用iTRAQ技术研究周围神经感觉和运动束Wallerian变性前后的差异表达蛋白

发布时间：2019-01-02 18:46

【摘要】：目的：通过构建大鼠周围神经损伤模型,比较感觉和运动神经束Wallerian变性前后差异表达蛋白,筛选感觉和运动神经束特异表达蛋白为周围神经趋化性再生机制的研究提供依据。方法：选择成年雄性8周龄Wistar大鼠60只,分成3组,其中A、B两组分别行股神经横断模型(n=20)和神经根损伤模型(n=20)的建立,C组作为神经根损伤模型的正常对照组(n=20),股神经横断模型选择对侧正常神经作为对照。7d后各组分别进行取材,并应用iTRAQ(isobaric tags for relative and absolute quantitation,同位素标记相对和绝对定量技术)联合LC-MS/MS (liquid chromatography-tandem mass spectrometry,液相色谱-串联质谱)对各组样本提取的蛋白进行同位素标记、质谱分析及差异蛋白的鉴定,应用DAVID (DAVID Bioinformatics Resources)对所鉴定出的差异蛋白进行GO(Gene ontology)功能和KEGG Pathway通路的富集,比较Wallerian变性后感觉和运动神经的差异表达蛋白,筛选与周围神经趋化性再生机制相关的蛋白。结果：(1)在股神经损伤模型中,共鉴定出3358个差异表达蛋白,其中股神经肌支Wallerian变性前后共鉴定出293个差异蛋白,隐神经变性前后共鉴定出417个差异蛋白,通过交集分析,肌支变性前后特异的差异蛋白有107个,隐神经变性前后特异的差异蛋白有231个。(2)在神经根损伤模型中,共鉴定出4312个差异表达蛋白,腹侧神经根变性前后共鉴定出637个差异蛋白,背侧神经根变性前后共鉴定出626个差异蛋白,通过交集分析,腹侧神经根变性前后特异的差异蛋白有195个,背侧神经根变性前后特异的差异蛋白有184个。结论：应用iTRAQ技术检测大鼠感觉和运动神经纤维变性前后表达差异蛋白,筛选感觉和运动神经变性后特异表达的蛋白,为周围神经趋化性再生的机制研究提供了依据。
[Abstract]:Aim: to establish a rat model of peripheral nerve injury, compare the differentially expressed proteins between sensory and motor tract Wallerian before and after denaturation, and screen the specific expression proteins of sensory and motor nerve bundles to provide evidence for the study of the mechanism of peripheral nerve chemotaxis regeneration. Methods: sixty adult male 8-week-old Wistar rats were randomly divided into 3 groups. The femoral nerve transection model (nong20) and the nerve root injury model (nnt20) were established in group A and B, respectively. Group C was used as the normal control group of nerve root injury model (NN20), and the contralateral normal nerve was selected as control in the model of femoral nerve transection. After 7 days, the samples were taken from each group and iTRAQ (isobaric tags for relative and absolute quantitation, was used. The relative and absolute quantitative techniques of isotopic labeling combined with LC-MS/MS (liquid chromatography-tandem mass spectrometry, liquid Chromatography-tandem Mass Spectrometry) were used to label the proteins extracted from each group of samples by isotope labeling, mass spectrometry analysis and differential protein identification. DAVID (DAVID Bioinformatics Resources) was used to enrich the GO (Gene ontology) function and KEGG Pathway pathway of the identified differentially expressed proteins, to compare the differentially expressed proteins of sensory and motor nerves after Wallerian degeneration, and to screen the proteins related to the mechanism of chemotaxis regeneration of peripheral nerve. Results: (1) in the model of femoral nerve injury, 3358 differentially expressed proteins were identified, including 293 differentially expressed proteins before and after denaturation of femoral nerve muscle branch and 417 differential proteins before and after degeneration of saphenous nerve. There were 107 differentially expressed proteins before and after muscular branch degeneration and 231 differential proteins before and after saphenous nerve degeneration. (2) 4312 differentially expressed proteins were identified in the model of nerve root injury. 637 differential proteins were identified before and after ventral nerve root degeneration, and 626 differential proteins were identified before and after dorsal nerve root degeneration. There were 184 specific differential proteins before and after dorsal nerve root degeneration. Conclusion: using iTRAQ technique to detect the differential protein expression before and after sensory and motor nerve fiber degeneration in rats, and to screen the specific protein expression after sensory and motor nerve degeneration, which provides the basis for the study of the mechanism of peripheral nerve chemotaxis regeneration.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.3

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