脊髓缺血再灌注损伤大鼠miRNA表达谱的筛选与初步分析

发布时间：2018-05-18 18:51

本文选题：脊髓缺血再灌注损伤 + miRNA　；参考：《吉林大学》2015年博士论文

【摘要】：脊髓损伤是一种很难治愈、后果严重的中枢神经系统损伤。学者们对脊髓损伤的病因及机制进行了大量的临床与基础研究。人们发现，除原发性脊髓损伤外，原发性脊髓损伤之后的继发性损害，如脊髓缺血再灌注损伤（spinal cordischemia-reperfusion injury，SCIRI）同样是造成脊髓神经系统损伤的一个重要因素。然而，针对于脊髓缺血再灌注损伤目前尚缺乏有效、公认的治疗药物和治疗手段。鉴于此，揭示脊髓缺血再灌注损伤的病理分子机制迫在眉睫。 microRNA（miRNA）是由18~24个核苷酸分子组成的短链非编码RNA分子，miRNA是一类具有强大功能的代谢调节分子，可以特异性抑制mRNA的翻译过程，进而在转录后水平调节基因的表达。最近的研究表明，miRNA存在于包括脑、脊髓在内的哺乳动物中枢神经系统（central nervous system，CNS）中，而且在中枢神经系统损伤、损伤修复以及变性疾病的病理生理进程中起到了重要的调节作用。但是，miRNA在缺血再灌注损伤的受损脊髓组织中的调节模式目前尚不明确，探究miRNA在脊髓缺血再灌注损伤病理生理进程以及损伤修复过程中的调控模式，对推动脊髓缺血再灌注损伤治疗手段的进步以及针对脊髓缺血再灌注损伤的特效药物研发具有重大意义。因此，本研究中我们构建了脊髓缺血再灌注大鼠模型，并利用Qiagen公司的miRCURYTM LNA Array(v.16.0) miRNA芯片分别检测脊髓单纯缺血损伤、脊髓缺血再灌注损伤后大鼠脊髓组织中的miRNA表达谱，建立差异表达miRNA谱（大于1.5倍）。并在利用实时定量PCR实验方法在脊髓缺血再灌注损伤模型大鼠脊髓组织中验证miRNA芯片中得到特殊差异表达miRNA。随后利用microRNA.org、Microcosm、miRBase数据库对差异表达倍数大于5倍的差异表达miRNA的靶基因进行预测，并对预测的靶基因进行基因本体论（GO）以及pathway富集分析；同时在TRED数据库中提取靶基因中的转录因子信息，利用Cytoscape vesion3.2.0软件绘制了miRNAs-TFs-Genes调控网络图，初步分析这些高差异倍数miRNA所调控的生物学功能。研究目的基于大鼠脊髓缺血再灌注损伤模型，高通量筛选基于大鼠脊髓缺血再灌注损伤区域组织中异常表达的miRNA，并通过以这些异常表达miRNA为核心构建分子调控网络，，旨在从整体角度对大鼠脊髓缺血再灌注损伤模型中miRNA的调控模式进行初步探讨。研究方法一、大鼠脊髓缺血再灌注损伤模型构建通过阻断成熟SD大鼠的腹主动脉构建脊髓缺血再灌注损伤大鼠模型，将24只SD大鼠分为：假手术组、单纯脊髓缺血组、脊髓缺血再灌注24小时组以及脊髓缺血再灌注48小时组。利用Basso Beattie Bresnahan（BBB）评分系统对脊髓缺血再灌注后大鼠的运动神经功能进行评价。利用Hematoxylin-Eosin（HE）染色实验方法检测脊髓缺血再灌注损伤后脊髓神经细胞形态的变化情况。并统计分析，确定大鼠脊髓缺血再灌注损伤模型成功构建。二、脊髓缺血再灌注损伤大鼠miRNA表达谱筛选提取大鼠脊髓缺血再灌注损伤模型中脊髓组织总RNA，利用miRCURYTMLNA Array(v.16.0) miRNA芯片分别检测单纯脊髓缺血组、脊髓缺血再灌注24小时组以及脊髓缺血再灌注48小时组差异表达miRNA谱，利用实时定量PCR实验验证miRNA芯片的表达数据。利用非监督性层次聚类方法分析差异表达miRNA信息。三、脊髓缺血再灌注损伤大鼠miRNA调控模式初步分析合并脊髓缺血再灌注24小时组和脊髓缺血再灌注48小时组的差异表达miRNA数据作为脊髓缺血再灌注损伤整体差异表达miRNA模式，选取差异表达倍数大于等于5倍的miRNA，利用microRNA.org、Microcosm、miRBase获取这些miRNA的靶基因信息，选取交集为miRNA的靶基因预测结果，利用DAVID以及webgestalt数据库对靶基因进行GO分析以及pathway富集分析。然后利用TRED数据库获取大鼠转录因子（Transcriptional Factor，TF）数据，并利用Cytoscape vesion3.2.0软件绘制miRNA调控网络。研究结果一、大鼠脊髓缺血再灌注损伤模型构建 1、手术组大鼠较假手术组大鼠下肢功能明显降低，再灌注48小时内，功能逐渐好转，再灌注24小时内功能改善程度最为明显。 2、神经元数目随再灌注时间延长逐渐减少，再灌注48小时最为稀少；间质水肿呈先加重后减轻变化，再灌注24小最明显，再灌注48小时较前减轻。 3、本研究成功构建了脊髓缺血再灌注损伤大鼠模型。二、脊髓缺血再灌注损伤大鼠miRNA表达谱筛选 1、在单纯缺血组、脊髓缺血再灌注组（24小时、48小时）中差异表达miRNA数目分别为115个（39个上调，76个下调）、13个（12个上调，1个下调）、105个（44个上调，61个下调）。 2、在单纯缺血组、脊髓缺血再灌注组（24小时、48小时）中，rno-miR-22-3p持续上调表达，且在单纯缺血组中上调倍数最高。 3、层次聚类分析结果显示，差异表达miRNA可准确的对单纯缺血组和脊髓缺血再灌注组（24小时、48小时）进行分类。三、脊髓缺血再灌注损伤大鼠miRNA调控模式初步分析 1、利用生物信息学手段，构建了脊髓单纯缺血组、脊髓缺血再灌注组、两种脊髓损伤共有的以及脊髓缺血再灌注损伤独有的miRNAs-TFs-Genes网络调控模式。 2、脊髓缺血再灌注损伤与脊髓单纯缺血损伤共享部分miRNA构成的调控网络，调控了细胞增殖与死亡调控中两个重要的转录因子Sp4和Cebpb。 3、脊髓缺血再灌注损伤独有的miRNA调控模式可能会通过调节MAPK信号转导通路，参与细胞增殖、分化、存活与凋亡，进而影响脊髓缺血再灌注损伤的发生与发展过程。 4、rno-miR-22-3p可能通过Tp53等转录因子调控细胞凋亡。研究结论 1、差异表达miRNA可准确的区分大鼠脊髓缺血再灌注损伤组与假手术组。 2、脊髓缺血再灌注损伤与脊髓单缺血损伤共享部分miRNA调控网络，其可能通过Sp4和Cebpb两个重要的转录因子调控细胞的增殖与死亡。 3、脊髓缺血再灌注损伤独有的miRNA调控模式可能会通过调节MAPK信号转导通路，参与细胞增殖、分化、存活与凋亡，进而影响脊髓缺血再灌注损伤的发生与发展过程。 4、rno-miR-22-3p在脊髓缺血再灌注损伤大鼠中可能通过Tp53等转录因子调控细胞凋亡。创新点 1、以系统生物学的理念，运用生物信息学的手段初步探讨了大鼠缺血再灌注损伤模型中miRNA调控模式。 2、首次在大鼠缺血再灌注损伤模型中报道了miR-22的作用与功能。
[Abstract]:Spinal cord injury (SCI) is a very difficult, serious and consequence central nervous system injury. A large number of clinical and basic studies have been conducted on the etiology and mechanism of spinal cord injury. It is found that secondary damage after primary spinal cord injury, such as spinal cordischemia-reper, is a secondary damage to primary spinal cord injury (such as spinal cord ischemia reperfusion injury). Fusion injury, SCIRI) is also an important factor in the injury of spinal cord nervous system. However, it is still lack of effective, recognized therapeutic drugs and treatment methods for spinal cord ischemia reperfusion injury. In view of this, the pathological molecular mechanism of spinal cord ischemia reperfusion injury is imminent.
MicroRNA (miRNA) is a short chain non coded RNA molecule composed of 18~24 nucleotides. MiRNA is a class of metabolic regulators with powerful functions that can specifically inhibit the translation of mRNA and then regulate the expression of genes at post transcriptional levels. Recent studies have shown that miRNA exists in the mammalian center, including the brain and spinal cord. In central nervous system (CNS), it plays an important role in the central nervous system injury, injury repair and the pathophysiological process of degenerative diseases. However, miRNA is not clear before the order of regulation in the damaged spinal cord tissue of ischemia-reperfusion injury, to explore the injury of miRNA in the spinal cord ischemia reperfusion injury. Pathophysiological processes and regulatory patterns in the process of injury repair are of great significance for progress in promoting the treatment of spinal cord ischemia-reperfusion injury and on the research and development of specific drugs for the spinal cord ischemia reperfusion injury.
Therefore, in this study, we constructed a rat model of spinal cord ischemia and reperfusion, and used the miRCURYTM LNA Array (v.16.0) miRNA chip of Qiagen company to detect the miRNA expression profiles in spinal cord tissue of rats after spinal cord ischemia reperfusion injury, and establish the differential expression of miRNA spectrum (more than 1.5 times). PCR test method was used in spinal cord ischemia reperfusion injury model rat spinal cord tissue to verify the special difference expression in the miRNA chip. Then, the target gene of the differential expression miRNA was predicted by the microRNA.org, Microcosm, miRBase database, and the gene ontology of the predicted target gene (G) was used for the Gene Ontology (G) for the predicted target gene (G). O) and pathway enrichment analysis; at the same time, the transcriptional factor information in the target gene was extracted from the TRED database, and the Cytoscape vesion3.2.0 software was used to plot the miRNAs-TFs-Genes regulation network map, and the biological functions regulated by these high differential miRNA were preliminarily analyzed.
research objective
Based on the model of rat spinal cord ischemia reperfusion injury, the abnormal expression of miRNA in the regional tissue of rat spinal cord ischemia reperfusion injury is screened by high throughput screening, and the molecular regulatory network is constructed by using these abnormal expression miRNA as the core. The aim of this study is to carry out the control model of miRNA in the rat model of spinal cord ischemia reperfusion injury from the whole point of view. Preliminary discussion.
research method
Construction of rat model of spinal cord ischemia reperfusion injury
The rat model of spinal cord ischemia reperfusion injury was constructed by blocking the abdominal aorta of mature SD rats. 24 SD rats were divided into three groups: sham operation group, simple spinal cord ischemia group, 24 hour group of spinal cord ischemia reperfusion and 48 hours of spinal cord ischemia reperfusion. The Basso Beattie Bresnahan (BBB) scoring system was used for the rats after the spinal cord ischemia and reperfusion. The function of motor nerve was evaluated. The changes of spinal nerve cell morphology after spinal cord ischemia reperfusion injury were detected by Hematoxylin-Eosin (HE) staining, and the model of spinal cord ischemia reperfusion injury was successfully constructed.
Two, miRNA expression profile in rats with spinal cord ischemia-reperfusion injury.
The total RNA of spinal cord tissue was extracted from rat spinal cord ischemia reperfusion injury model. MiRCURYTMLNA Array (v.16.0) miRNA chip was used to detect simple spinal cord ischemia, 24 hour group of spinal cord ischemia and reperfusion and 48 hour group of spinal cord ischemia and reperfusion were used to express miRNA spectrum. The expression data of miRNA chip were verified by real time quantitative PCR test. The unsupervised hierarchical clustering method was used to analyze the differential expression of miRNA information.
Three, a preliminary analysis of miRNA regulation in rats with spinal cord ischemia-reperfusion injury.
The differential expression of miRNA data in a 24 hour group of spinal cord ischemia reperfusion and 48 hours of spinal cord ischemia reperfusion was expressed as a miRNA model for the overall difference in the spinal cord ischemia reperfusion injury, and the differential expression multiplier was more than 5 times more than miRNA, and microRNA.org, Microcosm, miRBase were used to obtain the target gene information of these miRNA, and the intersection was selected as M. The target gene of iRNA was predicted by using the DAVID and webgestalt database for GO analysis and pathway enrichment analysis. Then the TRED database was used to obtain the rat transcription factor (Transcriptional Factor, TF) data, and Cytoscape vesion3.2.0 software was used to draw the miRNA regulation network.
Research results
Construction of rat model of spinal cord ischemia reperfusion injury
1, the lower limb function of the rats in the operation group was significantly lower than that of the sham operation group. The function gradually improved within 48 hours, and the function improvement was the most obvious within 24 hours of reperfusion.
2, the number of neurons decreased gradually with the prolongation of reperfusion time, and the most scarce in 48 hours of reperfusion; the interstitial edema was first aggravated and then reduced, and 24 of the reperfusion was the most obvious, and the reperfusion was reduced for 48 hours.
3, a rat model of spinal cord ischemia-reperfusion injury was successfully constructed.
Two, miRNA expression profile in rats with spinal cord ischemia-reperfusion injury.
1, in the simple ischemic group, the number of differential expressions of miRNA in the spinal cord ischemia reperfusion group (24 hours, 48 hours) was 115 (39 up-regulated, 76 down-regulated), 13 (12 up-regulated, 1 down-regulated), 105 (44 up-regulated, 61 down-regulation).
2, in the ischemic group, rno-miR-22-3p was continuously up-regulated in the spinal cord ischemia-reperfusion group (24 hours, 48 hours), and it was the highest in the ischemic group.
3, hierarchical clustering analysis showed that differential expression of miRNA could accurately classify the ischemic group and the spinal cord ischemia-reperfusion group (24 hours, 48 hours).
Three, a preliminary analysis of miRNA regulation in rats with spinal cord ischemia-reperfusion injury.
1, using bioinformatics, the miRNAs-TFs-Genes network regulation model of spinal cord ischemia and reperfusion group, spinal cord ischemia reperfusion group, two spinal cord injury and spinal cord ischemia reperfusion injury were constructed.
2, the regulatory network of miRNA, a shared part of spinal cord ischemia reperfusion injury and spinal cord ischemia injury, regulates the two important transcription factors, Sp4 and Cebpb., in cell proliferation and death regulation.
3, the unique miRNA regulation pattern of spinal cord ischemia reperfusion injury may be involved in cell proliferation, differentiation, survival and apoptosis by regulating the MAPK signal transduction pathway, and then affects the occurrence and development of spinal cord ischemia reperfusion injury.
4, rno-miR-22-3p may regulate apoptosis through transcription factors such as Tp53.
research conclusion
1, differential expression of miRNA can accurately distinguish rat spinal cord ischemia-reperfusion injury group and sham operation group.
2, spinal cord ischemia reperfusion injury and spinal single ischemic injury share part of the miRNA regulatory network, which may regulate cell proliferation and death through two important transcription factors of Sp4 and Cebpb.
3, the unique miRNA regulation pattern of spinal cord ischemia reperfusion injury may be involved in cell proliferation, differentiation, survival and apoptosis by regulating the MAPK signal transduction pathway, and then affects the occurrence and development of spinal cord ischemia reperfusion injury.
4, rno-miR-22-3p may regulate apoptosis through Tp53 and other transcription factors in rats with spinal cord ischemia-reperfusion injury.
innovation point
1, based on the concept of system biology, the miRNA regulation mode in rat models of ischemia-reperfusion injury was preliminarily discussed by means of bioinformatics.
2, for the first time, the role and function of miR-22 were reported in rat models of ischemia-reperfusion injury.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.2

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