创伤失血性休克大鼠血清及心肌miRNA标志物筛选及机理研究

发布时间：2018-06-23 07:18

本文选题：创伤失血性休克 + miRNA　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：目的创伤失血性休克(Traumatic hemorrhagic shock, THS)作为临床常见疾病之一,以其发病机制复杂、病程长及死亡率高等特点已被广泛关注,多种细胞炎症因子参与该过程的调控,但具体机制尚未完全阐明,特别是miRNA参与该过程调控的具体机制尚需进一步阐明。因此,本文拟采用急性机械性损伤方法建立大鼠THS模型,结合血清细胞因子和心肌巨噬细胞标志蛋白检测确定最佳检测时间点,给予miRNA二代测序和生物信息学分析得到THS大鼠血清和心肌表达差异miRNAs,结合miRNAs生物学功能验证以期获得其参与THS调控的具体机制,为从miRNA角度诠释THS的预防及治疗提供新的手段和潜在靶标。方法首先,基于急性机械性损伤法建立大鼠THS模型,并于造模后0h、1h、2h、 4h、8h、16h、24h、48h收集大鼠血清和心肌组织样本,采用酶联免疫吸附测定法检测血清肿瘤坏死因子-a(Tumor necrosis factor-alpha,TNF-α)、白细胞介素-2(Interleukin-2, IL-2)、白细胞介素-6(Interleukin-6, IL-6)、白细胞介素-10(Interleukin-10, IL-10)等细胞炎症因子表达水平以确定血清miRNA样本最佳检测时间点：针对心肌巨噬细胞MO、M1和M2型分化标志蛋白CD68,诱导性一氧化氮合酶(inducible nitric oxide synthase, iNOS)和精氨酸酶-1(Arginase-1, ARG1)利用免疫组织化学(Immunohistochemistry, IHC);去检测以确定心肌miRNA样本最佳检测时间点。然后,针对THS大鼠血清采用Illumina HiSeq4000系统进行小RNA(small RNA, sRNA)深度测序,通过生物信息学分析获得血清差异性表达miRNAs及聚类,结合Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集获得rniRNA调控的靶基因生物学功能及信号通路。针对血清样本中显著上调的5个miRNAs和显著下调的5个miRNAs,采用实时定量PCR(qRT-PCR)验证,选择已确证存在显著差异的miR-92a-1-3p和细胞因子进行相关性分析。最后,针对THS大鼠心肌组织采用Illumina HiSeq4000系统进行sRNA深度测序,通过生物信息学分析获得血清差异性表达miRNAs及聚类,结合Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集获得获得miRNA调控的靶基因生物学功能及信号通路。针对与Toll样受体-4(Toll-like receptors, TLR-4)调控相关的5个心肌细胞miRNAs,采用qRT-PCR验证,选择已确证存在显著差异的miR-155采用双荧光素酶报告基因系统验证其对靶基因TLR4的调控,进而研究心肌细胞因子TNF-α转化生长因子-β(transforming growth factor-β, TGF-β)、IL-6、IL-10的表达变化。同时,针对]miR-155 mimic进行体外合成,尾静脉注射后结合苏木精-伊红(Hematoxylin-eosin, HE)染色和IHC检测心肌组织病理学变化及巨噬细胞M1和M2型标志蛋白。结果1)利用急性机械性损伤法成功建立了大鼠THS模型,炎性因子TNF-α和IL-6的表达水平随着造模时间的增加显著降低,4h达谷值并缓慢升高至稳定期,抗炎因子IL-2和IL-10的表达水平随造模时间的增加显著增加,4h达峰值并缓慢降低至稳定期,说明4h是血清的最佳变化时间点。同样地,大鼠心肌组织巨噬细胞MO型、M1型和M2型极化标志蛋白CD68、iNOS和ARG1在造模后16h变化最明显,说明16h是心肌细胞最佳变化时间点。2)血清测序结果显示：共获得86个显著差异miRNAs,包括68个已知miRNAs和18个新miRNAs。其中,miR-92a-1-3p的表达水平与高通量测序分析结果一致,并且与细胞因子IL-6和CRP负相关,与细胞因子IL-10正相关。3)心肌组织测序结果显示：共发现744个miRNAs,包括729个已知miRNAs和15个新miRNAs。其中,miR-155的表达水平与高通量测序分析结果一致,可通过调控TLR4的3'-UTR去调控其转录和翻译。外源miR-155 mimic可显著抑制巨噬细胞M1型极化标志蛋白iNOS及其分泌的炎性细胞因子TNF-α和IL-6的表达,促进巨噬细胞M2型极化标志蛋白ARG1及其分泌的炎性细胞因子IL-10和MCL1的表达。结论本研究通过高通量测序、生物信息学分析及分子生物学验证获得了血清及心肌组织特异性差异表达的miRNA,为THS相关疾病的治疗提供了新的方案和靶标。同时,从miRNA角度解释其可通过干预TLR4影响巨噬细胞M1型和M2型极化状态,为THS致心肌细胞损伤作用机理研究提供了重要参考,具有一定的临床应用价值。
[Abstract]:Objective Traumatic hemorrhagic shock (THS) is one of the common clinical diseases. It has been widely concerned with its complicated pathogenesis, long course of disease and high mortality, and many cytokines are involved in the regulation of the process, but the specific mechanism has not been fully elucidated, especially the specific mechanism of miRNA involved in the regulation of the process. The system still needs to be further clarified. Therefore, this paper uses acute mechanical damage method to establish the rat THS model, combined with serum cytokine and myocardial macrophage marker protein detection to determine the best detection time point, and give miRNA two generation sequencing and bioinformatics analysis to obtain the difference of serum and myocardial expression between THS rats and miRNAs, combined with miRNAs birth. In order to obtain the specific mechanism to participate in the regulation of THS, it provides new means and potential targets for the prevention and treatment of THS from the miRNA point of view. Method first, based on the acute mechanical damage method, the rat THS model was established. After the model, 0h, 1H, 2h, 4h, 8h, 16h, 24h, 48h rats serum and myocardial tissue samples were collected and enzymes were used. The level of serum tumor necrosis factor -a (Tumor necrosis factor-alpha, TNF- alpha), interleukin -2 (Interleukin-2, IL-2), interleukin -6 (Interleukin-6, IL-6), interleukin -10 (IL-2) and other cytokines were detected to determine the optimal detection time point of serum samples: needle MO, M1 and M2 type differentiation marker protein CD68, inducible nitric oxide synthase (inducible nitric oxide synthase, iNOS) and arginase -1 (Arginase-1, ARG1) were used to determine the optimum detection time point of the cardiac muscle samples. The Illumina HiSeq4000 system was used to sequence the small RNA (small RNA, sRNA) depth, and the serum differential expression miRNAs and clustering were obtained through bioinformatics analysis. The biological function and signal pathway of the target gene were enriched with Gene Ontology (GO) and Kyoto Encyclopedia. 5 miRNAs and 5 miRNAs, which were significantly up-regulated, were verified by real-time quantitative PCR (qRT-PCR), and the correlation analysis of miR-92a-1-3p and cytokine, which had proved significant differences, was selected. Finally, the Illumina HiSeq4000 system was used for THS rat myocardial tissue to be sequenced by sRNA depth, and the blood was obtained by bioinformatics analysis. Differentially expressed miRNAs and clustering, combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment to obtain the biological functions and signaling pathways of the target genes regulated by miRNA. The selection was confirmed for 5 cardiac cells. MiR-155 with a significant difference was used to verify the regulation of the target gene TLR4 by the dual luciferase reporter gene system, and then study the expression changes of TNF- alpha transforming growth factor - beta (transforming growth factor- beta, TGF- beta), IL-6, IL-10. At the same time, it was synthesized in vitro for]miR-155 mimic, combined with the tail vein injection. Hematoxylin eosin (Hematoxylin-eosin, HE) staining and IHC detection of myocardial histopathological changes and macrophage M1 and M2 type marker proteins. Results 1) rat THS model was successfully established by acute mechanical injury. The expression level of inflammatory factors TNF- A and IL-6 decreased significantly with the increase of model time, and the value of 4H reached the Valley and increased slowly. The expression level of anti inflammatory factors IL-2 and IL-10 increased significantly with the increase of model time, and 4H reached the peak and slowed down to the stable period, indicating that 4H was the best time point for the change of serum. Similarly, the MO, M1 and M2 polarization markers of macrophage in rat myocardium were marked by egg white CD68, iNOS and ARG1 were most obvious after the model was made. 16h was the best time point.2 of cardiac myocytes.) serum sequencing results showed that 86 significant differences were obtained, including 68 known miRNAs and 18 new miRNAs., the expression level of miR-92a-1-3p was consistent with high throughput sequencing analysis, and negative correlation with cytokine IL-6 and CRP, and the positive correlation with cytokine IL-10.3) myocardium group The results of sequencing showed that 744 miRNAs, including 729 known miRNAs and 15 new miRNAs. were found. The expression level of miR-155 was consistent with the high throughput sequencing analysis, and the transcription and translation could be regulated by regulating the 3'-UTR of TLR4. Exogenous miR-155 mimic could significantly inhibit the M1 polarization marker protein iNOS and its secretion of macrophage cells. The expression of inflammatory cytokines TNF- alpha and IL-6 promotes the expression of macrophage M2 polarization marker protein ARG1 and its secreted inflammatory cytokines, IL-10 and MCL1. Conclusion this study obtained the miRNA of specific differential expression of serum and myocardial tissue by high throughput sequencing, bioinformatics analysis and molecular biological verification, which is a related disease of THS. The treatment of disease provides a new scheme and target. At the same time, it can be explained from the miRNA point of view that it can affect the M1 and M2 type polarization state of macrophages by interfering with TLR4. It provides an important reference for the study of the mechanism of myocardial injury induced by THS, and has a certain clinical value.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R605.971

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