基于RNA-Seq分析大鼠脊髓损伤局部基因转录水平的变化

发布时间：2018-03-31 15:48

本文选题：脊髓损伤　切入点：RNA-Seq技术　出处：《蚌埠医学院》2017年硕士论文

【摘要】：研究背景:脊髓损伤(Spinal cord injury,SCI)会导致运动和感觉功能完全丧失。尽管脊髓外科手术技术在不断进步,但对于这种棘手的神经系统损害仍然缺少有效的治疗方法。因此,进一步研究SCI的分子机制是非常重要的。在本研究中,我们采用RNA测序技术(RNA-Seq)对大鼠SCI后不同阶段的全基因组转录谱进行分析,以系统地鉴定SCI病理过程中的关键基因和信号通路。目的:1.阐明大鼠SCI局部基因转录水平表达的变化规律;2.筛选、鉴定出SCI病理过程中的关键分子和信号通路。方法:(1)采用纽约大学SCI仪制备重物坠落SCI大鼠模型;(2)采用Basso Beattie Bresnahan(BBB)运动功能评分观察各实验组SCI大鼠运动情况;(3)采用RNA-Seq技术分析SCI不同阶段损伤局部基因在转录水平的表达变化;(4)选择具有显著变化的基因,采用实时荧光定量逆转录聚合酶链反应(q RT-PCR)进行验证;(5)对差异表达的基因进行Gene ontology(GO)和Kyoto encyclopedia of genesand genomes(KEGG)等生物信息学分析;(6)采用Western Blot在蛋白水平进行验证;(7)采用免疫组织荧光染色法对所选蛋白进行细胞定位。结果:RNA-Seq分析表明:与假手术组比较,急性损伤组(SCI-1d)共有1797个差异表达的基因,其中1223个上调和574个下调;亚急性损伤组(SCI-6d)总共有6590个差异表达的基因,其中3460个上调和3130个下调;慢性损伤组(SCI-28d)组共有3499个差异表达的基因,其中1866个上调和1633个下调(通过DESeq调整后p0.05)。GO富集分析显示显著富集的基因主要为免疫反应、MHC蛋白复合物、抗原加工和递呈、翻译相关基因、核糖体的结构组成、离子通道活性、小GTPase介导的信号转导、细胞因子/趋化活性等。KEGG通路分析表明显著富集的途径包括核糖体、抗原加工和递呈、逆行内源性大麻素信号、轴突导向、多巴胺能神经突触、谷氨酸能突触、突触、TNF-α、HIF-1α、NF-κB、Toll样受体、NOD样受体、c AMP、钙、催产素、Rap1、B细胞受体和趋化因子信号通路。我们进一步选取两个高表达的分子Ncf1和Plau,在蛋白水平探讨它们在SCI后的表达规律和细胞定位。结果表明,SCI后二者表达均升高。Ncf1在亚急性期和慢性期高表达,而Plau整个损伤过程表达均升高。它们均分布于浸润的白细胞。结论:1.RNA-Seq分析表明:与假手术组比较,脊髓损伤大鼠损伤局部急性期有1797个差异基因;亚急性期有6590个差异基因;慢性期3499个差异基因。2.在脊髓损伤急性期,核糖体、TNF-α、蛋白多糖、疟疾和金黄色葡萄球菌感染等是富集的信号通路;亚急性期包括谷氨酸能突触、circadian entrainment、GABA能突触、逆行内源性大麻素信号等;慢性期有逆行内源性大麻素信号、催产素信号、Rap1信号、突触囊泡循环等。3.本实验在m RNA和蛋白水平进一步探讨了Ncf1和Plau在脊髓损伤后的表达和定位。结果证实脊髓损伤后二者表达均增加。Ncf1在亚急性期和慢性期高表达,而Plau在急性期、亚急性期和慢性期表达均升高。高表达的Ncf1和Plau均分布于浸润的白细胞,提示它们参与SCI的炎症过程。
[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.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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