本文選題：脊髓損傷　切入點(diǎn)：RNA-Seq技術(shù)　出處：《蚌埠醫(yī)學(xué)院》2017年碩士論文

【摘要】：研究背景:脊髓損傷(Spinal cord injury,SCI)會導(dǎo)致運(yùn)動和感覺功能完全喪失。盡管脊髓外科手術(shù)技術(shù)在不斷進(jìn)步,但對于這種棘手的神經(jīng)系統(tǒng)損害仍然缺少有效的治療方法。因此,進(jìn)一步研究SCI的分子機(jī)制是非常重要的。在本研究中,我們采用RNA測序技術(shù)(RNA-Seq)對大鼠SCI后不同階段的全基因組轉(zhuǎn)錄譜進(jìn)行分析,以系統(tǒng)地鑒定SCI病理過程中的關(guān)鍵基因和信號通路。目的:1.闡明大鼠SCI局部基因轉(zhuǎn)錄水平表達(dá)的變化規(guī)律;2.篩選、鑒定出SCI病理過程中的關(guān)鍵分子和信號通路。方法:(1)采用紐約大學(xué)SCI儀制備重物墜落SCI大鼠模型;(2)采用Basso Beattie Bresnahan(BBB)運(yùn)動功能評分觀察各實驗組SCI大鼠運(yùn)動情況;(3)采用RNA-Seq技術(shù)分析SCI不同階段損傷局部基因在轉(zhuǎn)錄水平的表達(dá)變化;(4)選擇具有顯著變化的基因,采用實時熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(q RT-PCR)進(jìn)行驗證;(5)對差異表達(dá)的基因進(jìn)行Gene ontology(GO)和Kyoto encyclopedia of genesand genomes(KEGG)等生物信息學(xué)分析;(6)采用Western Blot在蛋白水平進(jìn)行驗證;(7)采用免疫組織熒光染色法對所選蛋白進(jìn)行細(xì)胞定位。結(jié)果:RNA-Seq分析表明:與假手術(shù)組比較,急性損傷組(SCI-1d)共有1797個差異表達(dá)的基因,其中1223個上調(diào)和574個下調(diào);亞急性損傷組(SCI-6d)總共有6590個差異表達(dá)的基因,其中3460個上調(diào)和3130個下調(diào);慢性損傷組(SCI-28d)組共有3499個差異表達(dá)的基因,其中1866個上調(diào)和1633個下調(diào)(通過DESeq調(diào)整后p0.05)。GO富集分析顯示顯著富集的基因主要為免疫反應(yīng)、MHC蛋白復(fù)合物、抗原加工和遞呈、翻譯相關(guān)基因、核糖體的結(jié)構(gòu)組成、離子通道活性、小GTPase介導(dǎo)的信號轉(zhuǎn)導(dǎo)、細(xì)胞因子/趨化活性等。KEGG通路分析表明顯著富集的途徑包括核糖體、抗原加工和遞呈、逆行內(nèi)源性大麻素信號、軸突導(dǎo)向、多巴胺能神經(jīng)突觸、谷氨酸能突觸、突觸、TNF-α、HIF-1α、NF-κB、Toll樣受體、NOD樣受體、c AMP、鈣、催產(chǎn)素、Rap1、B細(xì)胞受體和趨化因子信號通路。我們進(jìn)一步選取兩個高表達(dá)的分子Ncf1和Plau,在蛋白水平探討它們在SCI后的表達(dá)規(guī)律和細(xì)胞定位。結(jié)果表明,SCI后二者表達(dá)均升高。Ncf1在亞急性期和慢性期高表達(dá),而Plau整個損傷過程表達(dá)均升高。它們均分布于浸潤的白細(xì)胞。結(jié)論:1.RNA-Seq分析表明:與假手術(shù)組比較,脊髓損傷大鼠損傷局部急性期有1797個差異基因;亞急性期有6590個差異基因;慢性期3499個差異基因。2.在脊髓損傷急性期,核糖體、TNF-α、蛋白多糖、瘧疾和金黃色葡萄球菌感染等是富集的信號通路;亞急性期包括谷氨酸能突觸、circadian entrainment、GABA能突觸、逆行內(nèi)源性大麻素信號等;慢性期有逆行內(nèi)源性大麻素信號、催產(chǎn)素信號、Rap1信號、突觸囊泡循環(huán)等。3.本實驗在m RNA和蛋白水平進(jìn)一步探討了Ncf1和Plau在脊髓損傷后的表達(dá)和定位。結(jié)果證實脊髓損傷后二者表達(dá)均增加。Ncf1在亞急性期和慢性期高表達(dá),而Plau在急性期、亞急性期和慢性期表達(dá)均升高。高表達(dá)的Ncf1和Plau均分布于浸潤的白細(xì)胞,提示它們參與SCI的炎癥過程。
[Abstract]:Background: spinal cord injury (Spinal cord, injury, SCI) will lead to complete loss of sensation and motor function. Although the surgical technique for spinal cord surgery is in progress, but for the nervous system damage in this tough still lack of effective treatment. Therefore, further research on molecular mechanism of SCI is very important. In this study, we using RNA sequencing technology (RNA-Seq) on the whole genome transcription in different phases of the spectrum of rats after SCI analysis, the key to gene identification system in the pathogenesis of SCI and the signal pathway. Objective: 1. to clarify the expression of SCI in rat partial gene transcription; 2. screening, identify key molecules in the pathological process of SCI and the signal pathway. Methods: (1) preparing the heavy fall SCI rat model by New York University SCI instrument system; (2) using Basso Beattie Bresnahan (BBB) to observe the motor function scores of each experimental group SCI rats movement The situation; (3) the expression change of SCI in different stages of partial injury genes at the transcriptional level by RNA-Seq technology; (4) with significant changes in genes by real-time quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR) to verify; (5) Gene ontology of differentially expressed genes (GO) and Kyoto Encyclopedia of genesand genomes (KEGG) and bioinformatics analysis; (6) using Western Blot was verified at the protein level; (7) the location of the selected cell staining by immunohistochemistry. Results: RNA-Seq analysis showed that: compared with the sham operation group, acute injury group (SCI-1d) were 1797 the difference of gene, including 1223 up-regulated and 574 down regulated; sub acute injury group (SCI-6d) a total of 6590 differentially expressed genes, including 3460 up-regulated and 3130 down regulated; chronic injury group (SCI-28d group) a total of 3499 differentially expressed Of which 1866 genes were up-regulated and 1633 were down regulated (by DESeq adjusted P0.05).GO enrichment analysis showed that the main genes were significantly enriched for immune reaction, MHC protein complex, antigen processing and presentation, translation related genes, ribosomal structure composition, ion channel activity, signal transduction mediated by GTPase, cytokines / chemotactic activity.KEGG pathway analysis showed that significantly enriched pathways including ribosomes, antigen processing and presentation, retrograde endocannabinoid signaling, axon guidance, dopaminergic synapses and glutamatergic synapses, synaptic TNF-, alpha, alpha kappa HIF-1, NF- B, Toll like receptor, NOD like receptor, C AMP, calcium. Oxytocin, Rap1, B cell receptor and chemokine signaling pathway. We further selected two high expression of Ncf1 and Plau in molecular and protein levels of their expression pattern and SCI cells after positioning. The results showed that the expression of two were up SCI High.Ncf1 in subacute and chronic high expression, while Plau expression increased the damage process. They are distributed in the infiltration of white blood cells. Conclusion: 1.RNA-Seq analysis showed that: compared with the sham group, rats with spinal cord injury injury local acute 1797 genes; subacute 6590 differentially expressed genes 3499 genes; chronic phase.2. acute spinal cord injury, in the ribosome, TNF- alpha, proteoglycan, malaria and Staphylococcus aureus infection are the signal pathway enrichment; subacute stage including glutamatergic synapses, circadian entrainment, GABA synapses, retrograde endocannabinoid signaling; chronic retrograde endocannabinoid signal, oxytocin signal, Rap1 signal, synaptic vesicle cycle.3. the experiments at M and protein levels of RNA and Plau in Ncf1 to further explore the localization and expression after spinal cord injury. The results confirmed after spinal cord injury The two expression increased.Ncf1 expression in subacute and chronic phase, while Plau expression increased in acute phase, subacute phase and chronic phase. Ncf1 and Plau overexpressed in infiltrating white blood cells indicated that they were involved in SCI's inflammatory process.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.2

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本文編號：1691399