發(fā)布時(shí)間：2018-06-09 00:19

  本文選題：腦出血 + 腦皮層�。� 參考：《天津醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:腦出血是卒中的一種毀滅性的方式,在亞洲國家每年腦出血的發(fā)生率被發(fā)現(xiàn)以2-3倍增長。和白色人種相比中國人種被報(bào)道有更高的腦出血發(fā)生率。腦出血通常分為原發(fā)性和繼發(fā)性,在腦出血急性期,出血作為一個(gè)迅速擴(kuò)大的顱內(nèi)團(tuán)塊直接破壞神經(jīng)元的結(jié)構(gòu)和直接壓縮周圍的組織造成傷害,這個(gè)時(shí)期進(jìn)行及時(shí)處理非常關(guān)鍵,可以阻止組織的進(jìn)一步損害,而找到急性期預(yù)示病情變化的標(biāo)志物也成了關(guān)鍵點(diǎn)。目前對腦出血標(biāo)志物的研究進(jìn)行了很多,但是仍沒有確定的、被臨床認(rèn)可的標(biāo)記物。我們的研究基于以上的情況,對腦出血急性期各時(shí)間點(diǎn)的標(biāo)本進(jìn)行蛋白質(zhì)組學(xué)研究,獲得差異蛋白,并且通過western-blot進(jìn)行驗(yàn)證獲得標(biāo)記物蛋白及探索腦出血急性期可能的發(fā)病機(jī)制。方法:采用在立體定向儀下腦內(nèi)注入自體血(尾動(dòng)脈血50ul)構(gòu)造腦出血模型,分別在腦出血后2小時(shí)、4小時(shí)、6小時(shí)、8小時(shí)(每組5只大鼠)取血腫同側(cè)腦皮層組織及正常大鼠腦皮層組織進(jìn)行蛋白裂解(450μl 8M Urea和50μl的磷酸酶抑制劑裂解)、純度測定(Bradford法)、還原烷基化、液相預(yù)分離(RIGOL L-3000高效液相色譜儀)、質(zhì)譜測定(Thermo質(zhì)譜儀)、數(shù)據(jù)庫檢索(Proteome Discoverer1.4;搜索引擎:MASCOT;數(shù)據(jù)庫:NCBI-rat ref-sequence protein database(60122protein,updated on 07-2015)、統(tǒng)計(jì)學(xué)處理獲得差異蛋白。在獲得的差異蛋白中篩選出上調(diào)蛋白neurofibromin1和下調(diào)蛋白Ras-related protein Rab15,通過western-blot在同樣條件處理的組織樣品中驗(yàn)證其表達(dá)水平是否與質(zhì)譜篩選結(jié)果一致。結(jié)果:根據(jù)質(zhì)譜分析、數(shù)據(jù)庫檢索共獲得差異表達(dá)蛋白807個(gè),根據(jù)統(tǒng)計(jì)學(xué)處理與正常組對比具有明顯差異的上調(diào)蛋白169個(gè),下調(diào)蛋白173個(gè)。根據(jù)Ratio值及P值重點(diǎn)篩查出上調(diào)蛋白3個(gè):NF1、Vgf、Ncs1,下調(diào)蛋白8個(gè):Rab15、Slcla4、Eps15、Arhgap21、Npdc1、Mycbp、Nrn1、Tp53rk。選取上調(diào)蛋白neurofibromin1,下調(diào)蛋白Ras-related protein Rab15進(jìn)行western-blot,結(jié)果顯示腦出血后8小時(shí)內(nèi)蛋白neurofibromin1逐漸高表達(dá),而蛋白Ras-related protein Rab15是逐漸低表達(dá)。結(jié)論:1、腦出血急性期皮層神經(jīng)元蛋白表達(dá)有一定規(guī)律性,上調(diào)蛋白大部分是參與抑制調(diào)控的蛋白,下調(diào)蛋白大部分是參與蛋白轉(zhuǎn)運(yùn)、構(gòu)建和定位的蛋白;2、上調(diào)蛋白neurofibromin1,下調(diào)蛋白Ras-related protein Rab15可以作為腦出血急性期病情演變的標(biāo)記物或藥物治療靶點(diǎn);3、NF1-RAS通路可能是腦出血急性期的發(fā)病機(jī)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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R743.34

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相關(guān)期刊論文 前2條

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本文編號：1997899