本文選題：脊髓缺血再灌注損傷 + miRNA�。� 參考：《吉林大學(xué)》2015年博士論文

【摘要】：脊髓損傷是一種很難治愈、后果嚴(yán)重的中樞神經(jīng)系統(tǒng)損傷。學(xué)者們對(duì)脊髓損傷的病因及機(jī)制進(jìn)行了大量的臨床與基礎(chǔ)研究。人們發(fā)現(xiàn)，除原發(fā)性脊髓損傷外，原發(fā)性脊髓損傷之后的繼發(fā)性損害，如脊髓缺血再灌注損傷（spinal cordischemia-reperfusion injury，SCIRI）同樣是造成脊髓神經(jīng)系統(tǒng)損傷的一個(gè)重要因素。然而，針對(duì)于脊髓缺血再灌注損傷目前尚缺乏有效、公認(rèn)的治療藥物和治療手段。鑒于此，揭示脊髓缺血再灌注損傷的病理分子機(jī)制迫在眉睫。 microRNA（miRNA）是由18~24個(gè)核苷酸分子組成的短鏈非編碼RNA分子，miRNA是一類具有強(qiáng)大功能的代謝調(diào)節(jié)分子，可以特異性抑制mRNA的翻譯過程，進(jìn)而在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。最近的研究表明，miRNA存在于包括腦、脊髓在內(nèi)的哺乳動(dòng)物中樞神經(jīng)系統(tǒng)（central nervous system，CNS）中，而且在中樞神經(jīng)系統(tǒng)損傷、損傷修復(fù)以及變性疾病的病理生理進(jìn)程中起到了重要的調(diào)節(jié)作用。但是，miRNA在缺血再灌注損傷的受損脊髓組織中的調(diào)節(jié)模式目前尚不明確，探究miRNA在脊髓缺血再灌注損傷病理生理進(jìn)程以及損傷修復(fù)過程中的調(diào)控模式，對(duì)推動(dòng)脊髓缺血再灌注損傷治療手段的進(jìn)步以及針對(duì)脊髓缺血再灌注損傷的特效藥物研發(fā)具有重大意義。 因此，本研究中我們構(gòu)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芯片分別檢測脊髓單純?nèi)毖獡p傷、脊髓缺血再灌注損傷后大鼠脊髓組織中的miRNA表達(dá)譜，建立差異表達(dá)miRNA譜（大于1.5倍）。并在利用實(shí)時(shí)定量PCR實(shí)驗(yàn)方法在脊髓缺血再灌注損傷模型大鼠脊髓組織中驗(yàn)證miRNA芯片中得到特殊差異表達(dá)miRNA。隨后利用microRNA.org、Microcosm、miRBase數(shù)據(jù)庫對(duì)差異表達(dá)倍數(shù)大于5倍的差異表達(dá)miRNA的靶基因進(jìn)行預(yù)測，并對(duì)預(yù)測的靶基因進(jìn)行基因本體論（GO）以及pathway富集分析；同時(shí)在TRED數(shù)據(jù)庫中提取靶基因中的轉(zhuǎn)錄因子信息，利用Cytoscape vesion3.2.0軟件繪制了miRNAs-TFs-Genes調(diào)控網(wǎng)絡(luò)圖，初步分析這些高差異倍數(shù)miRNA所調(diào)控的生物學(xué)功能。 研究目的 基于大鼠脊髓缺血再灌注損傷模型，高通量篩選基于大鼠脊髓缺血再灌注損傷區(qū)域組織中異常表達(dá)的miRNA，并通過以這些異常表達(dá)miRNA為核心構(gòu)建分子調(diào)控網(wǎng)絡(luò)，，旨在從整體角度對(duì)大鼠脊髓缺血再灌注損傷模型中miRNA的調(diào)控模式進(jìn)行初步探討。 研究方法 一、大鼠脊髓缺血再灌注損傷模型構(gòu)建 通過阻斷成熟SD大鼠的腹主動(dòng)脈構(gòu)建脊髓缺血再灌注損傷大鼠模型，將24只SD大鼠分為：假手術(shù)組、單純脊髓缺血組、脊髓缺血再灌注24小時(shí)組以及脊髓缺血再灌注48小時(shí)組。利用Basso Beattie Bresnahan（BBB）評(píng)分系統(tǒng)對(duì)脊髓缺血再灌注后大鼠的運(yùn)動(dòng)神經(jīng)功能進(jìn)行評(píng)價(jià)。利用Hematoxylin-Eosin（HE）染色實(shí)驗(yàn)方法檢測脊髓缺血再灌注損傷后脊髓神經(jīng)細(xì)胞形態(tài)的變化情況。并統(tǒng)計(jì)分析，確定大鼠脊髓缺血再灌注損傷模型成功構(gòu)建。 二、脊髓缺血再灌注損傷大鼠miRNA表達(dá)譜篩選 提取大鼠脊髓缺血再灌注損傷模型中脊髓組織總RNA，利用miRCURYTMLNA Array(v.16.0) miRNA芯片分別檢測單純脊髓缺血組、脊髓缺血再灌注24小時(shí)組以及脊髓缺血再灌注48小時(shí)組差異表達(dá)miRNA譜，利用實(shí)時(shí)定量PCR實(shí)驗(yàn)驗(yàn)證miRNA芯片的表達(dá)數(shù)據(jù)。利用非監(jiān)督性層次聚類方法分析差異表達(dá)miRNA信息。 三、脊髓缺血再灌注損傷大鼠miRNA調(diào)控模式初步分析 合并脊髓缺血再灌注24小時(shí)組和脊髓缺血再灌注48小時(shí)組的差異表達(dá)miRNA數(shù)據(jù)作為脊髓缺血再灌注損傷整體差異表達(dá)miRNA模式，選取差異表達(dá)倍數(shù)大于等于5倍的miRNA，利用microRNA.org、Microcosm、miRBase獲取這些miRNA的靶基因信息，選取交集為miRNA的靶基因預(yù)測結(jié)果，利用DAVID以及webgestalt數(shù)據(jù)庫對(duì)靶基因進(jìn)行GO分析以及pathway富集分析。然后利用TRED數(shù)據(jù)庫獲取大鼠轉(zhuǎn)錄因子（Transcriptional Factor，TF）數(shù)據(jù)，并利用Cytoscape vesion3.2.0軟件繪制miRNA調(diào)控網(wǎng)絡(luò)。 研究結(jié)果 一、大鼠脊髓缺血再灌注損傷模型構(gòu)建 1、手術(shù)組大鼠較假手術(shù)組大鼠下肢功能明顯降低，再灌注48小時(shí)內(nèi)，功能逐漸好轉(zhuǎn)，再灌注24小時(shí)內(nèi)功能改善程度最為明顯。 2、神經(jīng)元數(shù)目隨再灌注時(shí)間延長逐漸減少，再灌注48小時(shí)最為稀少；間質(zhì)水腫呈先加重后減輕變化，再灌注24小最明顯，再灌注48小時(shí)較前減輕。 3、本研究成功構(gòu)建了脊髓缺血再灌注損傷大鼠模型。 二、脊髓缺血再灌注損傷大鼠miRNA表達(dá)譜篩選 1、在單純?nèi)毖M、脊髓缺血再灌注組（24小時(shí)、48小時(shí)）中差異表達(dá)miRNA數(shù)目分別為115個(gè)（39個(gè)上調(diào)，76個(gè)下調(diào)）、13個(gè)（12個(gè)上調(diào)，1個(gè)下調(diào)）、105個(gè)（44個(gè)上調(diào)，61個(gè)下調(diào)）。 2、在單純?nèi)毖M、脊髓缺血再灌注組（24小時(shí)、48小時(shí)）中，rno-miR-22-3p持續(xù)上調(diào)表達(dá)，且在單純?nèi)毖M中上調(diào)倍數(shù)最高。 3、層次聚類分析結(jié)果顯示，差異表達(dá)miRNA可準(zhǔn)確的對(duì)單純?nèi)毖M和脊髓缺血再灌注組（24小時(shí)、48小時(shí)）進(jìn)行分類。 三、脊髓缺血再灌注損傷大鼠miRNA調(diào)控模式初步分析 1、利用生物信息學(xué)手段，構(gòu)建了脊髓單純?nèi)毖M、脊髓缺血再灌注組、兩種脊髓損傷共有的以及脊髓缺血再灌注損傷獨(dú)有的miRNAs-TFs-Genes網(wǎng)絡(luò)調(diào)控模式。 2、脊髓缺血再灌注損傷與脊髓單純?nèi)毖獡p傷共享部分miRNA構(gòu)成的調(diào)控網(wǎng)絡(luò)，調(diào)控了細(xì)胞增殖與死亡調(diào)控中兩個(gè)重要的轉(zhuǎn)錄因子Sp4和Cebpb。 3、脊髓缺血再灌注損傷獨(dú)有的miRNA調(diào)控模式可能會(huì)通過調(diào)節(jié)MAPK信號(hào)轉(zhuǎn)導(dǎo)通路，參與細(xì)胞增殖、分化、存活與凋亡，進(jìn)而影響脊髓缺血再灌注損傷的發(fā)生與發(fā)展過程。 4、rno-miR-22-3p可能通過Tp53等轉(zhuǎn)錄因子調(diào)控細(xì)胞凋亡。 研究結(jié)論 1、差異表達(dá)miRNA可準(zhǔn)確的區(qū)分大鼠脊髓缺血再灌注損傷組與假手術(shù)組。 2、脊髓缺血再灌注損傷與脊髓單缺血損傷共享部分miRNA調(diào)控網(wǎng)絡(luò)，其可能通過Sp4和Cebpb兩個(gè)重要的轉(zhuǎn)錄因子調(diào)控細(xì)胞的增殖與死亡。 3、脊髓缺血再灌注損傷獨(dú)有的miRNA調(diào)控模式可能會(huì)通過調(diào)節(jié)MAPK信號(hào)轉(zhuǎn)導(dǎo)通路，參與細(xì)胞增殖、分化、存活與凋亡，進(jìn)而影響脊髓缺血再灌注損傷的發(fā)生與發(fā)展過程。 4、rno-miR-22-3p在脊髓缺血再灌注損傷大鼠中可能通過Tp53等轉(zhuǎn)錄因子調(diào)控細(xì)胞凋亡。 創(chuàng)新點(diǎn) 1、以系統(tǒng)生物學(xué)的理念，運(yùn)用生物信息學(xué)的手段初步探討了大鼠缺血再灌注損傷模型中miRNA調(diào)控模式。 2、首次在大鼠缺血再灌注損傷模型中報(bào)道了miR-22的作用與功能。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.2

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相關(guān)期刊論文 前1條

1 蔣萌;王霖;蔣海河;;脊髓內(nèi)MAPK-ERK通路在心肌缺血再灌注損傷中的作用[J];中國當(dāng)代兒科雜志;2013年05期

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