脑出血急性期脑皮层蛋白质组学研究

发布时间：2018-06-09 00:19

本文选题：脑出血 + 脑皮层　；参考：《天津医科大学》2016年博士论文

【摘要】：目的:脑出血是卒中的一种毁灭性的方式,在亚洲国家每年脑出血的发生率被发现以2-3倍增长。和白色人种相比中国人种被报道有更高的脑出血发生率。脑出血通常分为原发性和继发性,在脑出血急性期,出血作为一个迅速扩大的颅内团块直接破坏神经元的结构和直接压缩周围的组织造成伤害,这个时期进行及时处理非常关键,可以阻止组织的进一步损害,而找到急性期预示病情变化的标志物也成了关键点。目前对脑出血标志物的研究进行了很多,但是仍没有确定的、被临床认可的标记物。我们的研究基于以上的情况,对脑出血急性期各时间点的标本进行蛋白质组学研究,获得差异蛋白,并且通过western-blot进行验证获得标记物蛋白及探索脑出血急性期可能的发病机制。方法:采用在立体定向仪下脑内注入自体血(尾动脉血50ul)构造脑出血模型,分别在脑出血后2小时、4小时、6小时、8小时(每组5只大鼠)取血肿同侧脑皮层组织及正常大鼠脑皮层组织进行蛋白裂解(450μl 8M Urea和50μl的磷酸酶抑制剂裂解)、纯度测定(Bradford法)、还原烷基化、液相预分离(RIGOL L-3000高效液相色谱仪)、质谱测定(Thermo质谱仪)、数据库检索(Proteome Discoverer1.4;搜索引擎:MASCOT;数据库:NCBI-rat ref-sequence protein database(60122protein,updated on 07-2015)、统计学处理获得差异蛋白。在获得的差异蛋白中筛选出上调蛋白neurofibromin1和下调蛋白Ras-related protein Rab15,通过western-blot在同样条件处理的组织样品中验证其表达水平是否与质谱筛选结果一致。结果:根据质谱分析、数据库检索共获得差异表达蛋白807个,根据统计学处理与正常组对比具有明显差异的上调蛋白169个,下调蛋白173个。根据Ratio值及P值重点筛查出上调蛋白3个:NF1、Vgf、Ncs1,下调蛋白8个:Rab15、Slcla4、Eps15、Arhgap21、Npdc1、Mycbp、Nrn1、Tp53rk。选取上调蛋白neurofibromin1,下调蛋白Ras-related protein Rab15进行western-blot,结果显示脑出血后8小时内蛋白neurofibromin1逐渐高表达,而蛋白Ras-related protein Rab15是逐渐低表达。结论:1、脑出血急性期皮层神经元蛋白表达有一定规律性,上调蛋白大部分是参与抑制调控的蛋白,下调蛋白大部分是参与蛋白转运、构建和定位的蛋白;2、上调蛋白neurofibromin1,下调蛋白Ras-related protein Rab15可以作为脑出血急性期病情演变的标记物或药物治疗靶点;3、NF1-RAS通路可能是脑出血急性期的发病机制之一。
[Abstract]:Objective: intracerebral hemorrhage (ICH) is a devastating form of stroke and the annual incidence of ICH has been found to increase 2-3 times in Asian countries. Chinese people are reported to have a higher incidence of intracerebral hemorrhage than white people. Intracerebral hemorrhage is usually divided into primary and secondary. In the acute phase of intracerebral hemorrhage, as a rapidly expanding mass of intracranial mass, directly destroys the structure of neurons and directly compresses the surrounding tissues. It is critical to deal with this period in a timely manner to prevent further tissue damage and to find markers that predict changes in the acute phase. Many studies on ICH markers have been carried out, but there is no identified clinically recognized marker. Based on the above situation, we studied the proteomics of the samples at different time points in the acute stage of intracerebral hemorrhage, obtained the differential protein, and obtained the marker protein by western-blot, and explored the possible pathogenesis of the acute stage of intracerebral hemorrhage. Methods: intracerebral hemorrhage model was established by injecting autologous blood (caudal blood 50ul) into the brain under stereotactic instrument. The hematomas in the ipsilateral cortex of the hematoma and the normal cortical tissue of the brain were taken at 2 hours, 4 hours, 6 hours and 8 hours after intracerebral hemorrhage, respectively. The protein cleavage of 450 渭 l M Urea and 50 渭 l of phosphatase inhibitor cleavage were carried out in the same side of the hematoma and in the normal cerebral cortex of the rats. Determination of Bradford method for reductive alkylation, RIGOL L-3000 high performance liquid chromatograph (HPLC), Thermo mass spectrometer (MS), Proteome Discovery 1.4; search engine: MacOT; Database: NCBI-rat ref-sequence protein database 60122 protein updated on 07-2015.The difference protein was obtained by statistical analysis. The up-regulated protein neurofibromin1 and the down-regulated Ras-related protein Rab15 were screened out from the differentially obtained proteins. The expression level of western-blot in tissue samples treated with the same conditions was confirmed to be consistent with the results of mass spectrometry. Results: according to mass spectrometry, 807 differentially expressed proteins were obtained by database retrieval. 169 up-regulated proteins and 173 down-regulated proteins were compared with normal controls by statistical analysis. According to the ratio value and P value, the up-regulation protein 3: NF1 / VgfU Ncs1 and down-regulated protein 8: Rab15 / Slcla4EPS15 / Arhgap21 / Npdc1 / MycbpNrn1 / Tp53rkwere selected. The up-regulation protein neurofibromin 1 and down-regulation protein Ras-related protein Rab15 were selected for western-blot. The results showed that the protein neurofibromin1 expression gradually increased within 8 hours after intracerebral hemorrhage, while the protein Ras-related protein Rab15 was gradually low expression. Conclusion the expression of protein in cortical neurons is regular in the acute phase of intracerebral hemorrhage. Most of up-regulated proteins are involved in inhibition and regulation, and down-regulated proteins are mainly involved in protein transport. Construction and localization of protein 2, up-regulation of protein neurofibromin1 and down-regulation of Ras-related protein Rab15 may be used as markers or drug therapy targets for the progression of acute stage of intracerebral hemorrhage (ICH), which may be one of the pathogenetic mechanisms of ICH in acute stage.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.34

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