【作者】 刘志刚；

【导师】 杨小玉；

【作者基本信息】 吉林大学 ， 外科学， 2015， 博士

【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。更多还原

【Abstract】 Spinal cord ischemia-reperfusion injury (SCIRI) is an important pathogenicmechanism of spinal cord secondary injury. Studies show that the secondary injury,such as SCIRI, is an important factor involved in nerve injury. However, there are noeffective treatments and specific drugs towards SCIRI, and the pathogenic molecularmechanism of SCIRI is still unclear now, which are big hindrances to thedevelopments of novel effective treatments and specific drugs. Hence, it is extremelyurgent to underline the molecular mechanism of SCIRI.MicroRNA (or miRNA) is an endogenous non-coding18~24nt short chain RNAmolecule, which has specific mRNA translation inhibitory function, that wouldregulate the gene expression at post-transcriptional level. Recent studies show that,numbers of miRNAs exist in mammalian central nervous system (CNS), includingbrain and spinal cord, where miRNAs were involved in the regulation of CNS injury,repair following the injury and neurotraumatic pathologies. But, the regulatoryfunctions of miRNAs in spinal cord are still unclear. So, study the miRNA regulatorypattern in lesion region of SCIRI would clarify the molecular mechanism of SCIRI,which has great significance on the development of efficient treatments and novelspecific drugs.In our study, we successfully constructed a SCIRI Rat model, and utilizedmiRCURYTM LNA Array (V.16.0) to analyze the miRNA expression profiles oflesion region of spinal cord ischemia injury and SCIRI. QRT-PCR was used to verifythe data of miRNA microarray. Target gene prediction of the differentially expressedmiRNAs (Fold charge over5) was performed based on the data collected from severaldatabases, including microRNA.org、Microcosm、miRBase. Following the prediction,GO analysis and pathway enrichment analysis were performed to identify thebiological function of these differentially expressed miRNAs. Additionally,miRNAs-TFs-Genes regulatory networks were built to visualize the regulatory patternof miRNAs. PurposesBased on SCIRI rat model, high-throughout screen the differentially expressedmiRNAs in lesion tissues. And through the construction of miRNAs-TFs-Genesregulatory networks to gain preliminary analysis of the miRNA regulatory pattern inSCIRI rat model, from a global point of view.Methods1. Construction of SCIRI rat modelSCIRI rat model was induced in mature SD rats by occluding the descendingthoracic aorta. The24mature SD rats were assigned randomly into four groups: shamoperation group; spinal cord ischemia injury group; spinal cord ischemia-reperfusioninjury group (24-hour group and48-hour group). Neurological functions of SCIRI ratmodel were assessed at24and48hour after reperfusion. Then spinal cords wereharvested for hematoxylin-eosin (HE) staining and verified that the SCIRI rat modelwas constructed successfully.2. Screening of rat SCIRI miRNA expression profileWe extracted the total RNA from spinal cord of SCIRI rat model, and utilizedmiRCURYMLNA Array (V.16.0) to detect the miRNA expression profile of spinalcord ischemia injury group and spinal cord ischemia-reperfusion injury groups(24-hour group and48-hour group). QRT-PCR was then used to verify the miRNAexpression profile. Unsupervised hierarchical clustering was performed to analyze themiRNA expression profile and finally we performed the bioinformatics analysis toidentify the biological functions of differentially expressed miRNAs.3. Preliminary analysis of miRNA regulatory pattern in SCIRI rat modelWe combined the miRNA expression profile data of two spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group) as a totalmiRNA expression profile of SCIRI rat model. MiRNAs of which fold charge was noless than5were collected for significant miRNA regulatory pattern analysis. Targetprediction of these selected differentially expressed miRNA was performed based onthe data provided in public databases including MicroRNA.org, Microcosm andMiRanda. Then, databases, including DAVID and webgestalt, were utilized toperform GO analysis and pathway enrichment analysis. Finally, intersection of abovethree databases was used for miRNAs-TFs-Genes regulatory networks construction,where the transcriptional factors (TFs) were identified based on the data provided by TRED database. Cytoscape software version3.2.0was used to visualize themiRNAs-TFs-Genes regulatory networks information.Results1. Construction of SCIRI rat model1.1Compared to control group, rats in operation group had significantly lowerlimb function, and would gradually improved in48hours, of which content is obviousin the first24hours.1.2HE staining of spinal cords showed that neurons were rare in spinal cords ofSCIRI rat model, which got worst at the48hour. And interstitial edema increasedgradually until the24hour, which remitted after reperfusion for48hours.1.3Results in this study proved that SCIRI rat model was successfullyconstructed for further studies.2. Screening of rat SCIRI miRNA expression profile2.1115(39up regulated,76down regulated),13(12up regulated,1downregulated) and105（44up regulated,61down regulated) differentially expressedmiRNAs were respectively detected in spinal cord tissues of spinal cord ischemiainjury group and spinal cord ischemia-reperfusion injury groups (24-hour group and48-hour group).2.2Among spinal cord ischemia injury group and spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group),rno-miR-22-3p was steadily high-expressed, and reached the peak in spinal cordischemia injury group.2.3Unsupervised hierarchical clustering analysis showed that spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group) can bedistinguished accurately.3. Preliminary analysis of miRNA regulatory pattern in SCIRI rat model3.1We constructed miRNAs-TFs-Genes regulatory networks of spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group) bybioinformatics tools.3.2Spinal cord ischemia injury and SCIRI may share part of miRNA regulatorynetworks, which may regulate cell proliferation and death by transcriptional factorSp4and Cebpb.3.3The specific miRNA regulatory pattern of SCIRI may be involved in cell proliferation, differentiation, survival and death, through the regulation of MAPKsignaling pathway, which would play an important role in the progress of SCIRI.3.4Rno-mir-22-3p would have key roles in cell apoptosis regulatory pathway, bythe regulation of transcriptional factor Tp53.Conclusions1. These differentially expressed miRNAs can accurately identify the SCIRI ratmodel.2. Spinal cord ischemia injury and SCIRI may share part of miRNA regulatorynetworks, which may regulate cell proliferation and death by transcriptional factorSp4and Cebpb.3. The specific miRNA regulatory pattern of SCIRI may be involved in cellproliferation, differentiation, survival and death, through the regulation of MAPKsignaling pathway, which would play an important role in the progress of SCIRI.4. Bioinformatics analysis shows that rno-miR-22expression in SCIRI rat modelspinal cord tissues would play an injury repair role through the regulation of cellapoptosis regulatory pathway.Innovation points1. Based on the systemic biology theory, using bioinformatics method，wepreliminarily analyzed the miRNA regulatory pattern in SCIRI rat model, from aglobal point of view.2. We reported the function of miR-22in SCIRI rat model for the first time.更多还原