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目的分析斑马鱼神经元模型损伤给药治疗前后总蛋白差异,寻找养血清脑颗粒对斑马鱼神经元损伤预后的生物标记物及其作用机制。方法采用核素标记相对和绝对定量技术结合纳升液相色谱和串联质谱(Nano LC-MS/MS)对给药前后斑马鱼药效模型总蛋白进行分离并分析肽段,采用Proteinpilot 4.2软件对蛋白进行鉴定和定量分析,对获得的差异蛋白进行生物信息学分析,应用蛋白印迹法对可能的生物标记物进行验证。结果通过Nano LC-MS/MS分析鉴定出置信度>95%的蛋白质共1 933种,其中总差异表达蛋白质130种,模型组与模型给药组表达量上调≥1.5倍的蛋白质有9种,下调≤0.70倍的蛋白质有23种。通过GO分析显示,差异蛋白的分子功能主要集中在神经元离子通道的翻译和代谢过程;Western blot结果显示,与模型组相比,差异表达蛋白Ef1-α(elongation factor 1-alpha)和α-6-F(Na~+/K~+ transporting ATPase alpha 1 polypeptide)在给药后表达量下调,差异具有统计学意义(P<0.05),且与质谱结果一致。结论 iTRAQ联合Nano LC-MS/MS技术能高通量地筛选斑马鱼神经元损伤相关蛋白,初步阐述养血清脑颗粒对斑马鱼神经元损伤预后的作用机制。
OBJECTIVE: To analyze the differences of total proteins between pre-treatment and post-treatment of zebrafish neuron injury model and find the biomarker of Yangxue Qingnao Granule on the prognosis of zebrafish neurons and its mechanism. Methods The total protein of zebrafish efficacy model before and after administration was separated and analyzed by nuclide labeling and tandem mass spectrometry (Nano LC-MS / MS) Protein identification and quantitative analysis of the differential proteins obtained for bioinformatics analysis, the use of Western blotting to validate the possible biomarkers. Results A total of 1 933 proteins with 95% confidence level were identified by Nano LC-MS / MS. Among them, 130 proteins were differentially expressed, 9 proteins were up-regulated 1.5 times or more in model group and model group, There are 23 kinds of proteins that are down ≤0.70 times. The results of GO analysis showed that the molecular functions of the differential proteins mainly focused on the translation and metabolism of neuronal ion channels. Western blot results showed that compared with the model group, the expressions of Ef1-α and α- The expression of 6-F (Na ~ + / K + + ATPase alpha 1 polypeptide) was down-regulated after administration, with statistical significance (P <0.05). Conclusion iTRAQ combined with Nano LC-MS / MS can screen neuronal damage related proteins in zebrafish with high throughput and elucidate the mechanism of Yangxue Qingnao Granule on the prognosis of zebrafish neurons.